Constructs for delivery of threrapeutic agents to neuronal cells

ABSTRACT

A non-toxic polypeptide, for delivery of a therapeutic agent to a neuronal cell, comprises a binding domain that binds to the neuronal cell, and a translocation domain that translocates the therapeutic agent into the neuronal cell, wherein the translocation domain is not a H N  domain of a clostridial toxin and is not a fragment or derivative of a H N  domain of a clostridial toxin.

[0001] The present invention relates to constructs for delivering therapeutic substances to neuronal cells, to manufacture and use thereof, and in particular to constructs based on clostridial neurotoxins.

[0002] There are presently few effective treatments for major disorders of the central nervous system. Such disorders include neurodegenerative diseases, stroke, epilepsy, brain tumours, infections and HIV encephalopathy, and sufferers of these diseases far outnumber the morbidity of cancer and heart disease. The number of sufferers for CNS disorders such as stroke and the neurodegenerative diseases is set to grow, particularly in developed countries where the average age of the population is increasing. As our understanding of brain pharmacology increases and the underlying pathologies of diseases are elucidated, potential therapeutic strategies become apparent. All these treatments, however, face the formidable problem of efficient delivery of therapeutics to the various neuronal cell populations involved. Vectors which can effect efficient delivery to neuronal cells are thus required for a broad range of therapeutic substances, including drugs, enzymes, growth factors, therapeutic peptides and genes.

[0003] Ischemia/reperfusion injury induced by stroke or injury is one notable example in which rapid and efficient delivery of therapeutic agents would afford considerable benefit. Neurons injured by trauma or ischemia produce elevated levels of free oxygen radicals and release large amount of glutamate. These substances in high concentration are toxic to both neurons and surrounding cells which potentiate and amplify the damage process. Agents such as superoxide dismutase or glutamine synthetase which reduce the levels of these toxic substances have been shown to reduce neuronal cell death in a variety of in vitro and in vivo ischemia models (Gorovits et al. PNAS (1997) 94, 7024-7029; Francis et al. Experimental Neurology (1997) 146, 435-443; Lim et al. Ann. Thorac. Surg. (1986) 42, 282-286; Cuevas et al. Acta Anat. (1990) 137, 303-310). A major problem in the use of such therapies is the delivery of useful concentrations of the active agent to the site of trauma. Specific neuronal vectors could therefore play an important role in targeting such compounds to neuronal cells.

[0004] Peripheral nervous system disorders, such as motor neuron disease, are further examples of diseases which would benefit from the targeted delivery of therapeutic agents. Such therapies could take the form of drug delivery or DNA delivery via gene therapy strategies.

[0005] Gene therapy holds considerable promise for the treatment of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Most of the currently available viral and non-viral gene delivery vectors lack tissue specificity which reduces both their efficiency and safety of use. Suitable neuronal cell-specific targeting ligands are therefore required for a broad range of gene vectors to enable effective treatments for neuronal diseases to be developed.

[0006] The botulinum neurotoxins are a family of protein toxins whose primary site of action is the neuromuscular junction where they block the release of the transmitter acetylcholine. The action of these toxins on the peripheral nervous system of man and animals results in the syndrome botulism, which is characterised by widespread flaccid muscular paralysis (Shone (1986) in ‘Natural Toxicants in Foods’, Editor D. Watson, Ellis Harwood, UK). Each of the botulinum neurotoxins consists of two disulphide-linked subunits; a 100 kDa heavy subunit which plays a role in the initial binding and internalisation of the neurotoxin into the nerve ending (Dolly et. al. (1984) Nature, 307, 457-460) and a 50 kDa light subunit which acts intracellularly to block the exocytosis process (McInnes and Dolly (1990) Febs Lett., 261, 323-326; de Paiva and Dolly (1990) Febs Lett., 277, 171-174).

[0007] The clostridial neurotoxins are potent inhibitors of calcium-dependent neurotransmitter secretion in neuronal cells. They are currently considered to mediate this activity through a specific endoproteolytic cleavage of at least one of three vesicle or pre-synaptic membrane associated proteins VAMP, syntaxin or SNAP-25 which are central to the vesicle docking and membrane fusion events of neurotransmitter secretion. The neuronal cell targeting of tetanus and botulinum neurotoxins is considered to be a receptor mediated event following which the toxins become internalised and subsequently traffic to the appropriate intracellular compartment where they effect their endopeptidase activity.

[0008] Clostridial neurotoxins share a common architecture of a catalytic L-chain (LC, ca 50 kDa) disulphide linked to a receptor binding and translocating H-chain (HC, ca 100 kDa). The HC polypeptide is considered to comprise all or part of two distinct functional domains. The carboxy-terminal half of the HC, termed the H_(C) domain (ca 50 kDa), is involved in the high affinity, neurospecific binding of the neurotoxin to cell surface receptors on the target neuron, whilst the amino-terminal half, termed the H_(N) domain (ca 50 kDa), is considered to mediate the translocation of at least some portion of the neurotoxin across cellular membranes such that the functional activity of the LC is expressed within the target cell. The H_(N) domain also has the property, under conditions of low pH, of forming ion-permeable channels in lipid membranes, and this may in some manner relate to its translocation function. For botulinum neurotoxin type A (BoNT/A) these domains are considered to reside within amino acid residues 872-1296 for the H_(C), amino acid residues 449-871 for the H_(N) and residues 1-448 for the LC.

[0009] It is therefore possible to provide functional definitions of the domains within the neurotoxin molecule, as follows:—

[0010] (A) clostridial neurotoxin light chain:—

[0011] a metalloprotease exhibiting high substrate specificity for vesicle and/or plasma membrane associated proteins involved in the exocytotic process. In particular, it cleaves one or more of SNAP-25, VAMP (synaptobrevin/cellubrevin) and syntaxin.

[0012] (B) clostridial neurotoxin heavy chain H_(N) domain:—

[0013] a portion of the heavy chain which enables translocation of that portion of the neurotoxin molecule such that a functional expression of light chain activity occurs within a target cell.

[0014] the domain responsible for translocation of the endopeptidase activity, following binding of neurotoxin to its specific cell surface receptor via the binding domain, into the target cell.

[0015] the domain responsible for formation of ion-permeable pores in lipid membranes under conditions of low pH.

[0016] (c) clostridial neurotoxin heavy chain H_(C) domain:

[0017] a portion of the heavy chain which is responsible for binding of the native holotoxin to cell surface receptor(s) involved in the intoxicating action of clostridial toxin prior to internalisation of the toxin into the cell.

[0018] The identity of the cellular recognition markers for these toxins is currently not understood and no specific receptor species have yet been identified although Kozaki et al. have reported that synaptotagmin may be the receptor for botulinum neurotoxin type B. It is probable that each of the neurotoxins has a different receptor.

[0019] Tetanus toxin is structurally very similar to botulinum neurotoxins but its primary site of action is the central nervous system where it blocks the release of inhibitory neurotransmitters from central synapses (Renshaw cells).

[0020] Tetanus and the botulinum neurotoxins from most of the seven serotypes, together with their derived heavy chains, have been shown to bind a wide variety of neuronal cell types with high affinities in the nM range, e.g. botulinum type B neurotoxin (Evans et al. (1986) Eur. J. Biochem. 154, 409-416).

[0021] However, a major obstacle to the use of the native clostridial heavy chain fragments as delivery vectors is that their highly aggregated state in solution prevent their adequate diffusion into body tissue and hence reduces their efficiency as targeting vectors. A further significant problem with any proposed clinical use of native tetanus toxin fragments as neuronal targeting ligands for therapeutics is the existence of circulating antibodies to the toxin in the majority of the population who have been immunized against tetanus. The presence of these antibodies is likely to reduce the efficacy of constructs based on tetanus toxin fragments. Thus, clostridial neurotoxin fragments do not offer solutions to the problems identified.

[0022] The present invention is based upon the discovery of the practical difficulties in using clostridial neurotoxin-based therapeutic compositions, and the devising of modified polypeptides and hybrid polypeptides based on clostridial neurotoxin fragments that avoid the aforementioned drawbacks.

[0023] Accordingly, a first aspect of the invention provides a non-toxic polypeptide, for delivery of a therapeutic agent to a neuronal cell, comprising:—

[0024] a binding domain that binds to the neuronal cell, and

[0025] a translocation domain that translocates the therapeutic agent into the neuronal cell,

[0026] wherein the translocation domain is not a H_(N) domain of a clostridial neurotoxin and is not a fragment or derivative of a H_(N) domain of a clostridial toxin.

[0027] The binding domain is suitably comprised of or derived from clostridial heavy chain fragments or modified clostridial heavy chain fragments. As used herein, the term “modified clostridial heavy chain fragment” means a polypeptide fragment which retains similar biological functions to the corresponding heavy chain of a botulinum or tetanus neurotoxin but differs in its amino acid sequence and other properties compared to the corresponding heavy chain. The invention more specifically provides such constructs which are based on fragments derived from botulinum and tetanus neurotoxins.

[0028] In a further aspect, the invention also provides a polypeptide, for delivery of a therapeutic agent to a neuronal cell, comprising:—

[0029] a binding domain that binds to the neuronal cell, and

[0030] a translocation domain that translocates the therapeutic agent into the neuronal cell,

[0031] wherein the resulting polypeptide construct is non-aggregating.

[0032] Whether the construct is an aggregating one is usually apparent from a lack of solubility of the construct, and this may be seen upon simple visual inspection of the construct in aqueous media: non-aggregating domains result in constructs of the invention that are partially or preferably totally soluble whereas aggregating domains result in non-soluble aggregates of polypeptides having apparent sizes of many tens or even hundreds the size of a single polypeptide. Generally, the construct should be non-aggregating as measured by size on gel electrophoresis, and the size or apparent size of the construct measured should preferably be less than 5.0×10⁵ daltons, more preferably less than 1.5×10⁵ daltons, with the measuring being suitably carried out on native PAGE using physiological conditions.

[0033] A still further aspect of the invention provides a polypeptide, for delivery of a therapeutic agent to a neuronal cell, comprising:—

[0034] a binding domain that binds to the neuronal cell, and

[0035] a translocation domain that translocates the therapeutic agent into the neuronal cell,

[0036] wherein the translocation domain is selected from (1) a H_(N) domain of a diphtheria toxin, (2) a fragment or derivative of (1) that substantially retains the translocating activity of the H_(N) domain of a diphtheria toxin, (3) a fusogenic peptide, (4) a membrane disrupting peptide, (5) a H_(N) from botulinum toxin C₂ and (6) translocating fragments and derivatives of (3), (4) and (5).

[0037] It is to be noted that botulinum toxin C₂ is not a neurotoxin as it has no neuronal specificity, instead it is an enterotoxin and suitable for use in the invention to provide a non-aggregating translocation domain.

[0038] A yet further aspect of the invention provides a polypeptide, for delivery of a therapeutic agent to a neuronal cell, comprising:—

[0039] a binding domain that binds to the neuronal cell, and

[0040] a translocation domain that translocates the therapeutic agent into the neuronal cell,

[0041] wherein the polypeptide has reduced affinity to neutralising antibodies to tetanus toxin compared with the affinity to such antibodies of native tetanus toxin heavy chain.

[0042] The above aspects may singly or in any combination be exhibited by polypeptides of the invention and thus a typical preferred polypeptide of the invention (i) lacks the neurotoxic activities of botulinum and tetanus toxins, (ii) displays high affinity to neuronal cells corresponding to the affinity of a clostridial neurotoxin for those cells, (iii) contains a domain which can effect translocation across cell membranes, and (iv) occurs in a less aggregated state than the corresponding heavy chain from botulinum or tetanus toxin in physiological buffers.

[0043] A significant advantage of the polypeptides of the invention is their non-aggregated state, thus rendering them usable as soluble polypeptides where the prior art constructs were not and overcoming most if not all of the drawbacks of previous constructs based upon clostridial neurotoxins.

[0044] The polypeptides according to the invention generally include sequences from the H_(C) domains of the botulinum and tetanus neurotoxins and these are combined with functional domains from other proteins, such that the essential functions of the native heavy chain, binding to neuronal cells, is retained. Thus, for example, the H_(C) domain of botulinum type F neurotoxin is fused to the translocation domain derived from diphtheria toxin to give a modified clostridial heavy chain fragment. Surprisingly, such polypeptides are more useful as constructs for delivering substances to neuronal cells than are the native clostridial heavy chains.

[0045] Thus, according to a preferred aspect of the invention there is provided a polypeptide having an amino acid sequence comprising (a) a sub-sequence based on the H_(C) fragment of botulinum or tetanus neurotoxin, and (b) a sub-sequence based on a translocation domain, e.g. from diphtheria toxin, that is not derived from a clostridial neurotoxin, and wherein the said polypeptide (i) lacks the neurotoxin activities of botulinum and tetanus toxins, (ii) displays high affinity to neuronal cells, (iii) contains a domain which can effect translocation across cell membranes and (iv) occurs in a less aggregated state than the corresponding heavy chain of botulinum or tetanus toxin in physiological buffers.

[0046] The modified clostridial heavy chain is suitably produced by combining the binding domain (H_(C) domain) of a clostridial neurotoxin with a non-clostridial translocation domain. Thus, for example, a modified clostridial heavy chain fragment may be constructed from the translocation domain of diphtheria toxin (residues 194-386) fused to the H_(C) domain of a botulinum toxin (e.g. type F H_(C) fragment, residues 865-1278; type A H_(C) fragment, residues 872-1296).

[0047] In another embodiment of the invention, the modified clostridial heavy chain is produced by combining the H_(C) domain of a clostridial neurotoxin with a membrane disrupting peptide which functions as a translocation domain, suitably a viral peptide. Thus, for example, a modified clostridial heavy chain fragment may be constructed by combining the H_(C) domain of a botulinum toxin with a peptide based on influenza virus haemagglutinin HA2 (residues 1-23).

[0048] The polypeptides of the invention have properties which make them useful as neuronal targeting ligands; they are non-toxic and yet retain the specific, high affinity binding to neuronal cells displayed by the botulinum or tetanus toxins. Unlike the native clostridial heavy chains, however, the modified clostridial heavy chains occur in a less aggregated state in solution which improves their access to neuronal cells. The preferred constructs are soluble in aqueous solution, in contrast to the highly aggregated state of the prior art constructs.

[0049] In another aspect of the invention, there is provided a modified tetanus heavy chain fragment which, in addition to the properties of modified heavy chains defined above, has the added advantage in that it has reduced affinity to neutralizing antibodies, present as a result of anti-tetanus inoculation, compared to the native tetanus toxin heavy chain. The polypeptides according to this aspect of the invention generally include subsequences derived from the heavy chain of tetanus toxin (residues 458-1315) and from which epitopes responsible for the immunogenicity of tetanus toxin have optionally been reduced or removed. Thus, for example, it is desirable to eliminate immunogenic epitopes associated with H_(C) domain as well as that of the H_(N) domain. Although it is possible to eliminate epitopes by deleting small numbers of amino acids (e.g. less than 20 or preferably less than 10 amino acids), it has been found that epitopes associated with immunogenicity of tetanus toxin heavy chain can be reduced more rigorously by replacing a large number of amino acid residues (e.g. at least 100, at least 200 and preferably 400 or more residues) with amino acid sequences from other toxins.

[0050] Thus according to a preferred aspect of the invention related to modified tetanus heavy chains, there is provided a polypeptide having an amino sequence comprising (a) an H_(N) domain derived from a non-clostridial source (e.g. diphtheria toxin), (b) one or more subsequences derived from the sequence of a botulinum H_(C), and (c) one or more subsequences derived from the sequence of tetanus toxin H_(C), and wherein said polypeptide (i) lacks the neurotoxin activities of botulinum and tetanus toxins, (ii) displays high affinity to neuronal cells corresponding to the neuronal binding of tetanus neurotoxin, (iii) contains a domain which can effect translocation across cell membranes and (iv) has low affinity to neutralizing antibodies to tetanus toxin which are present as result of anti-tetanus inoculation.

[0051] This latter modified tetanus heavy chain fragment can be produced by combining the binding domain (H_(C) domain) of tetanus neurotoxin with a non-clostridial translocation domain. Thus, for example, a modified tetanus heavy chain fragment may be constructed from the translocation domain of diphtheria toxin (residues 194-386) fused to the H_(C) domain of a tetanus toxin (residues 865-1315).

[0052] In another embodiment of the invention the modified tetanus heavy chain is derived a non-clostridial translocation domain fused to the H_(C) domain of a botulinum toxin into which the minimal domains of tetanus toxin are inserted to confer tetanus toxin-like binding activity onto the resulting hybrid. Thus, for example a modified tetanus heavy chain may be constructed from the translocation domain of diphtheria toxin (residues 194-386) fused to the H_(C) domain of a botulinum type F fragment (residues 865-1278) in which residues 1097-1273 of the latter have been replaced by homologous sequences from tetanus toxin.

[0053] The modified tetanus heavy chains have properties which make them useful as neuronal targeting ligands; they are non-toxic and yet retain the specific, high affinity binding to neuronal cells displayed by tetanus toxin. Unlike native tetanus toxin binding fragments, however, the modified clostridial binding fragments have different immunogenic properties which makes them more useful clinically. Specifically, the different immunogenic properties of the modified clostridial binding fragments of the invention significantly reduce the problems caused by existing antibodies to native tetanus toxin sequences.

[0054] While the use of modified heavy chains based on botulinum neurotoxins as neuronal targeting ligands does not suffer from the problem of pre-existing circulating antibodies, tetanus toxin is unique amongst the clostridial toxins in that it has selectivity to inhibitory neurons (e.g. Renshaw cells) and as such the modified tetanus toxin heavy chains are valuable targeting ligands for this class of neuron. Tetanus toxin also has the property that it can retrograde transport from the peripheral to the central nervous system.

[0055] In another embodiment of the invention, the modified clostridial heavy chain fragment is fused to a linker peptide via the N-terminus of the translocation domain to which a polypeptide payload may be attached. An examples of such a linker peptide is the sequence CGLVPAGSGP (SEQ ID NO:1) which contains the thrombin protease cleavage site and a cysteine residue for disulphide bridge formation. Such a peptide linker allows production of a recombinant fusion protein comprising a polypeptide therapeutic molecule fused by the linker peptide to the N-terminus of the modified clostridial heavy chain fragment. The latter single chain fusion protein may then be treated with thrombin to give a dichain protein in which the polypeptide therapeutic is linked to the translocation domain of the modified clostridial heavy chain fragment by a disulphide link. In another example of a linker peptide in which the translocation domain does not contain a free cysteine residue near its C-terminus, such as is the case when the translocation domain is a fusogenic peptide, the linker peptide contains both cysteine residues required for the disulphide bridge. An example of the latter linker peptide is the amino acid sequence: CGLVPAGSGPSAGSSAC (SEQ ID NO:2).

[0056] In another embodiment of the invention, the modified clostridial heavy chain is linked to a polypeptide which may be an enzyme, growth factor, protein or peptide which has therapeutic benefits when delivered to neuronal cells. The polypeptide may be linked to the modified clostridial heavy chain by chemical means. Alternatively the polypeptide may be produced as a fusion protein linked to the modified clostridial binding fragment by recombinant technology using the linker peptides as described above. In such an example, the construct would contain the following components:—

[0057] a polypeptide therapeutic substance;

[0058] a linker peptide; and

[0059] a modified clostridial heavy chain

[0060] An example of a polypeptide therapeutic payload is superoxide dismutase.

[0061] In yet another embodiment of the invention, the modified clostridial heavy chain is linked directly or indirectly to DNA such that the construct is capable of delivering the DNA to neuronal cells, e.g. via the receptor for tetanus toxin. Such constructs have gene therapy applications and be used to switch on, or off, selected genes with the cell. The DNA may be contained within a liposome or be condensed via a peptide or protein. The modified clostridial heavy chain may be chemically linked to the protein that effects the DNA condensation by chemical coupling agents. Alternatively, the modified clostridial heavy chain may be produced as a fusion protein, by recombinant technology, with a peptide that can effect the condensation of DNA.

[0062] In yet another embodiment of the invention, the modified clostridial heavy chain fragment may be linked to a recombinant virus such that the modified virus has an altered tropism and is capable of transducing cells via the tetanus toxin receptor. Such a construct is of use to correct genetic defects within neuronal cells by switching on, or off, selected genes. The modified clostridial heavy chain fragment may be linked directly to the surface of the virus using chemical cross-linking agents. Alternatively the modified clostridial heavy chain fragment may be linked to the recombinant virus via an antibody which specifically bind to the virus. In this instance the modified clostridial binding fragment is chemically coupled to a polyclonal or monoclonal antibody which specifically recognizes a marker on the surface of the virus. A similar modified clostridial binding fragment-antibody fusion protein could be produced by recombinant technology in which the antibody component is a recombinant single chain antibody.

[0063] In yet another embodiment of the invention, the modified clostridial heavy chain fragment is linked to a drug release system such as a microparticle constructed from a suitable polymer, e.g. poly (lactide-co-glycolide), polyhydroxylalkonate, collagen, poly(divinyl-ether-comaleic anhydride, poly (styrene-co-maleic anhydride) or other polymer useful in such microparticles. The modified clostridial heavy chain fragment may be linked to the drug release system by covalent chemical coupling, or electrostatic or hydrophobic forces. The modified clostridial heavy chain fragment may also be encapsulated within the release vehicle together with the therapeutic payload provided that a portion of the modified clostridial binding fragment is exposed at the surface. Alternatively, the modified clostridial heavy chain fragment may be linked, at either the N- or C-terminal end, to a peptide or protein to facilitate coupling of the fragment to the drug release system.

[0064] Other strategies are known by which modified heavy chain binding fragments can be linked to range of therapeutic substances using a variety of established chemical cross-linking techniques, and a variety of fusion proteins can be produced containing a modified clostridial binding fragment and another polypeptide. Using these techniques a variety of substances can be targeted to neuronal cells using the modified clostridial binding fragments. Examples of possible uses of the modified clostridial binding fragments as neuronal delivery vectors are given in more detail below in Table 1.

[0065] Constructs of the invention may be introduced into either neuronal or non-neuronal tissue using methods known in the art. By subsequent specific binding to neuronal cell tissue, the targeted construct exerts its therapeutic effects. Ideally, the construct is injected near a site requiring therapeutic intervention.

[0066] The construct of the invention may be produced as a suspension, emulsion, solution or as a freeze dried powder depending on the application and properties of the therapeutic substance. The construct of the invention may be resuspended or diluted in a variety of pharmaceutically acceptable liquids depending on the application.

[0067] “Clostridial neurotoxin” means either tetanus neurotoxin or one of the seven botulinum neurotoxins, the latter being designated as serotypes A, B C₁, D, E, F or G.

[0068] “Modified clostridial heavy chain fragment” means a polypeptide fragment which binds to neuronal cell receptors in similar manner to a corresponding heavy chain derived from botulinum or tetanus toxins but differs in its amino acid sequence and properties compared to the corresponding fragment derived from tetanus toxin.

[0069] “Bind” in relation to the botulinum and tetanus heavy chain fragments, means the specific interaction between the clostridial fragment and one or more cell surface receptors or markers which results in localization of the binding fragment on the cell surface. In the case of the clostridial neurotoxins, the property of a fragment being able to ‘bind’ like a fragment of a given serotype can be demonstrated by competition between the ligand and the native toxin for its neuronal cell receptor.

[0070] “High affinity binding specific to neuronal cell corresponding to that of a clostridial neurotoxin” refers to the ability of a ligand to bind strongly to cell surface receptors of neuronal cells that are involved in specific binding of a given neurotoxin. The capacity of a given ligand to bind strongly to these cell surface receptors may be assessed using conventional competitive binding assays. In such assays radiolabelled clostridial neurotoxin is contacted with neuronal cells in the presence of various concentrations of non-radiolabelled ligands. The ligand mixture is incubated with the cells, at low temperature (0-3° C.) to prevent ligand internalization, during which competition between the radiolabelled clostridial neurotoxin and non-labelled ligand may occur. In such assays when the unlabelled ligand used is the same as that of the labelled neurotoxin, the radiolabelled clostridial neurotoxin will be displaced from the neuronal cell receptors as the concentration of non-labelled neurotoxin is increased. The competition curve obtained in this case will therefore be representative of the behaviour of a ligand which shows “high affinity binding specificity to neuronal cells corresponding to that of a clostridial neurotoxin”, as used herein.

[0071] “Translocation domain” means a domain or fragment of a protein which effects transport of itself and/or other proteins and substances across a membrane or lipid bilayer. The latter membrane may be that of an endosome where translocation will occur during the process of receptor-mediated endocytosis. Translocation domains can frequently be identified by the property of being able to form measurable pores in lipid membranes at low pH (Shone et al. Eur J. Biochem. 167, 175-180). Examples of translocation domains are set out in more detail below in FIG. 1. In the application, translocation domains are frequently referred to as “H_(N) domains”.

[0072] “Translocation” in relation to translocation domain, means the internalization events which occur after binding to the cell surface. These events lead to the transport of substances into the cytosol of neuronal cells.

[0073] “Therapeutic substances” or “agents” mean any substance, agent or mixture thereof, which, if delivered by the modified clostridial binding fragment, would be beneficial to the treatment of neuronal diseases. Examples of these include drugs, growth factors, enzymes, and DNA packaged in various forms (e.g. modified viruses, cationic liposomes, and condensed DNA).

[0074] Also provided in the present invention are methods of manufacture of the polypeptides of the invention by expressing in a host cell a nucleic acid encoding the polypeptide, and the use of a polypeptide or a composition according to the invention in the treatment of a disease state associated with neuronal cells.

[0075] The invention is now illustrated in the following specific embodiments and accompanied by drawings in which:—

[0076]FIG. 1 shows modified clostridial heavy chain fragments produced by recombinant technology as a fusion proteins;

[0077]FIG. 2 shows modified clostridial heavy chain fragments produced by recombinant technology; fusion proteins may contain one or more purification peptide tags to assist in the purification of the protein;

[0078] one or more protease cleavage sites may also be included to enable removal of the purification peptide tags; similar purification strategies may also be employed for modified clostridial binding fragments containing a translocation domain;

[0079]FIG. 3 shows linkage of a modified clostridial binding fragment to a therapeutic substance; the modified clostridial heavy chain contains a translocation domain which has a free thiol group (an example of translocation domain with this property is amino acid sequence 194-386 of diphtheria toxin), a free amino group on the therapeutic substance is modified with a cross-linking reagent (e.g. SPDP; Pierce & Warriner, UK Ltd.) which will subsequently allow conjugate formation using the free thiol present on the modified clostridial binding fragment;

[0080]FIG. 4 shows the formation of a conjugate between a modified clostridial heavy chain fragment and an oligonucleotide as described in Example 4;

[0081]FIG. 5 shows a strategy for producing a recombinant modified clostridial heavy chain as a fusion protein with a polypeptide therapeutic substance. The latter is fused to the modified clostridial heavy chain by a linker peptide. The linker peptide contains a unique protease cleavage site (e.g. that recognized by thrombin) and a cysteine residue. Examples of linker peptides are (a) CGLVPAGSGP; and (b) CGIEGRAPGP (SEQ ID NO:18). The cysteine residue forms a disulphide bridge with an another available cysteine residue on the translocation domain of the modified heavy chain fragment. If desirable, then by treatment with thrombin, a dichain product may be produced in which the polypeptide therapeutic substance is linked to the heavy chain via a disulphide bridge;

[0082]FIG. 6 shows a comparison of the binding of a modified heavy chain with that of the native neurotoxin to neuronal synaptic membranes, the modified heavy chain displaying the binding characteristics of tetanus neurotoxin as assessed by the method described in Example 7;

[0083]FIG. 7 shows the binding to neuronal membranes of a modified clostridial heavy chain based on the binding domain of botulinum type F neurotoxin; in this example, modified heavy chain contained the translocation (H_(N)) domain of diphtheria toxin and the binding (H_(c)) domain of type F neurotoxin; and

[0084]FIG. 8 shows a comparison of the molecular sizes, under non-denaturing conditions, of a modified clostridial heavy chain compared to a native heavy chain; the modified clostridial heavy chain (Diphtheria H_(N)-BoNT/F H_(C)) runs as a monomer of approximately 70 kDa while a native heavy chain (from BoNT/A) runs as an aggregate of >500 kDa.

[0085] In more detail, FIG. 1 shows examples of embodiments of the invention incorporating modified clostridial heavy chain fragments.

[0086] The binding domain is derived from sequences of the clostridial neurotoxins:—

[0087] (a) H_(C) domains, e.g.

[0088] BoNT/A residues 872-1296

[0089] BoNT/B residues 859-1291

[0090] BoNT/C residues 867-1291

[0091] BoNT/D residues 863-1276

[0092] BoNT/E residues 846-1252

[0093] BoNT/F residues 865-1278

[0094] BoNT/G residues 864-1297

[0095] Tetanus residues 880-1315

[0096] (b) Hybrid H_(C) domains, e.g.

[0097] hybrids of the H_(C) domain of BoNT/F and tetanus

[0098] (c) Truncated H_(C) domains

[0099] The translocation domain may be derived from a number of sources:

[0100] (a) Bacterial toxins, e.g. diphtheria toxin fragment B (residues 194-386)

[0101] (b) Viral fusogenic peptides, e.g. from influenza virus haemagglutinin HA-2

[0102] (c) Synthetic membrane disrupting peptides (e.g. Plank et al., J. Biol. Chem., 269, 12918-12924).

[0103]FIG. 2 shows examples of Recombinant Modified Clostridial Heavy Chain Fragment Fusion Proteins Showing Positions of Purification Peptide Tags and Specific Protease Cleavage Sites (by treatment with the appropriate protease, the purification peptide tags may be removed from the modified clostridial binding fragment).

[0104] Examples of purification peptides tags are:

[0105] His6

[0106] S peptide

[0107] T7 peptide

[0108] Calmodulin binding peptide

[0109] Maltose binding protein

[0110] Examples of specific protease cleavage sites are:

[0111] Thrombin

[0112] Enterokinase

[0113] Factor X

EXAMPLE 1

[0114] Preparation and Purification of a Recombinant Modified Clostridial Heavy Chain Fragments.

[0115] Standard molecular biology protocols were used for all genetic manipulations (e.g. Sambrook et al. 1989, Molecular Cloning a Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). An entirely synthetic gene encoding the H_(C) regions of botulinum toxin from C. botulinum type F (residues 865-1278) and tetanus toxin (residues 880-1315) were generated using Recursive PCR reactions (Prodromou & Pearl 1992, Protein Engineering, 5: 827-829) using self-priming oligonucleotides containing the desired sequence. The codon bias and GC/AT base ratio was adjusted for ease of expression in E. coli. Fragments were cloned sequentially into pLitmus 38 (New England Biolabs, Inc., Beverly, Mass.) to assemble the entire gene. Constructs for expression were sub-cloned into pMALc2 (NEB) replacing the BamH1-EcoR1 fragment. The ligation reactions were transformed into E. coli JM109 (Promega).

[0116] Plasmid DNA was amplified, purified and screened for the presence of the appropriate sequence (Ausubel et al. 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York). Gene constructions confirmed as possessing the correct sequences were then transformed into the expression host E. coli BL21 (DE3) (Studier & Moffatt 1986, Journal of Molecular Biology, 189: 113-130).

[0117] Additional sequences for adding affinity purification tags and one or more specific protease site for the subsequent removal of these affinity tags were also included in the reading frame of the gene products.

[0118] The recombinant proteins expressed in pMAL were produced with amino-terminal maltose-binding protein tags allowing proteins to be purified by affinity chromatography on amylose resin. Briefly, cultures of E. coli BL21 (DE3) pMALc2-H_(C) were grown in Terrific broth-ampicillin (100 μgml⁻¹)-kanamycin (30 μgml⁻¹) to an OD₆₀₀ nm of 2.5-3.8, and protein expression was induced by the addition of 1 mM IPTG for approximately 2 h. Cells were lysed by freeze/thaw followed by sonication, lysates cleared by centrifugation and supernatants loaded onto an amylose resin column and eluted with maltose. All buffers used were as specified by the manufacturer. Thrombin or factor Xa protease sites were included within the protein for subsequent removal of these purification tags.

[0119] Other coding sequences which enable expression of the desired protein would also be acceptable. Other tags or linking sites may also be incorporated into the sequence. Examples of some of these options are summarized in FIG. 2.

EXAMPLE 2

[0120] Production of a Modified Clostridial Heavy Chain Fragments.

[0121] Using the techniques described in Example 1, modified clostridial heavy chain fragments was constructed by fusing domains of the H_(c) fragments of either botulinum type F or tetanus neurotoxins with the translocation domain of diphtheria toxin. The amino acid sequences of examples are shown in SEQ ID NO:s 8-17, which also gives examples of modified tetanus heavy chains in which the H_(c) fragment is a hybrid of tetanus and botulinum type F neurotoxin.

EXAMPLE 3

[0122] Coupling of a Modified Clostridial Heavy Chain Fragment to a Protein or an Enzyme.

[0123] The polypeptide, protein or enzyme to be linked to the modified clostridial heavy chain fragment is first derivatized with a suitable cross-linking agent. Mn-Superoxide dismutase (SOD) was modified by treatment with a 15 molar excess of SPDP (Pierce) in 0.05M Hepes buffer pH 7.0 containing 0.15M NaCl for 60 min at 25° C. The excess SPDP was removed by dialysis against the same buffer At 4° C. for 16 h. The substituted SOD was then mixed in a 1:5 molar ration with modified clostridial heavy chain fragment fused to a translocation domain derived from diphtheria toxin (see FIG. 3) and incubated at 25° C. for 16 h. After incubation the SOD-modified clostridial binding fragment conjugate was purified by gel filtration chromatography on Sephadex G200.

EXAMPLE 4

[0124] Coupling of Modified Clostridial Heavy Chain Fragment to Condensed DNA.

[0125] Poly-L-lysine (M_(r) 1000-4000) (10 mg) to be used for the condensation of DNA was dissolved in 2 ml of 20 mM Hepes buffer pH 7.4 containing 0.15M NaCl (HBS). To this solution 0.6 mg of Sulpho-LC-SPDP (Pierce and Warriner, UK Ltd.) was added and the mixture incubated for 30 min at 25° C. The activated poly-L-lysine was then dialysed against HBS at 4° C. using a dialysis tubing of 1000 molecular weight cut-off and then diluted to 1 mg/ml using HBS.

[0126] Condensation of DNA was carried out in glass tubes. Purified plasmid DNA containing a gene encoding a therapeutic protein (or a reporter gene) under the control of a suitable promoter (e.g. CMV immediate early, or a neuronal-specific promoter e.g. neuron-specific enolase promoter) was made 1 mg/ml in HBS and added to glass tubes followed by the activated poly-L-lysine as prepared above. Activated poly-L-lysine is added in various proportions to the DNA (see Table 2) and incubated for 90 min at 25° C. TABLE 2 Condensation of DNA with activated poly-L-lysine. Sample no. DNA(μg) Activated Poly-L-lysine HBS 1 750 250 1500 2 1500 500 500 3 500 250 1750 4 1000 500 1000

[0127] After incubation the size of the condensed DNA particles was assessed using a Brookhaven BI90 particle sizer. The incubation conditions giving the highest proportion of condensed DNA particle of less than 100 nM in diameter was used to produce DNA-modified clostridial binding fragment conjugates. Modified clostridial heavy chain was dialysed against HBS.

[0128] The dialysed fragments (100 μg) was then added to 1 ml of condensed DNA and incubated for 18 h at 25° C. to from the modified clostridial binding protein-condensed DNA construct (see FIG. 4).

EXAMPLE 5

[0129] Delivery of DNA to a Neuronal Cells via the Modified Clostridial Heavy Chain Fragment Receptor.

[0130] Modified clostridial heavy chain-condensed DNA construct described in Example 4 was diluted with 2 ml MEM serum free medium. Growth media from NG108 grown in 12 well dished was removed and 1 ml of the diluted construct added and incubated for 2 h at 37° C. in the presence of 5% CO₂. Growth media (1 ml) was then added to each well and the incubation continued under the same conditions for 24-48 h. After this period the cell were examined.

[0131] In experiments were the condensed DNA contained a reporter gene encoding Green Fluorescent Protein, several of the cells showed visible expression of the reporter protein illustrating successful delivery of the DNA into the neuronal cell. Various control experiments were conducted to confirm the observed transfection in NG108 cells was receptor mediated:

[0132] Transfection of NG108 cells was found to be dependent on the presence of modified clostridial heavy chain fragment within conjugates (no transfection was observed with condensed particles DNA alone)

[0133] No transfection was observed in non-neuronal cells (Vero cells) using the heavy chain-DNA conjugates.

EXAMPLE 6

[0134] Preparation of Conjugates of Modified Clostridial Heavy Chain Fragment and Microparticles Consisting of Poly(lactide-co-glycolide).

[0135] 398 mg of poly (lactide co-glycolide) low internal viscosity (3000 MW)(Beohringer Mannheim,) was dissolved in 4 ml dichloromethane. This was homogenised at 2000 rpm for 150 seconds with 1 ml of buffer solution containing the therapeutic substance, such as an enzymes and/or drugs. In the case of Mn superoxide dismutase, 10 mg of the enzyme was dissolved in 10 mM Hepes buffer pH 8.0 containing 100 mM NaCl. The mixture was then added to 50 ml of 8% poly vinyl alcohol and emulsified at 2000 rpm for a further 150 seconds. The emulsion was poured into 300 ml of ultrapure distilled water at 37° C. and stirred for 30 min at 37° C. The microparticles were collected by centrifugation at 10000×g for 25 min at 20° C. and then resuspended in 300 ml water and centrifuged as above. This washing procedure was the repeated a further 4 times. After the final centrifugation the water supernatant fluid was removed and the microparticles freeze dried.

[0136] 2 mg of poly (lactide-co-glycolide) microparticles were re-suspended in 1 ml of activation buffer (01 .M MES buffer, pH 6.0 containing 0.5M NaCl). Solid 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and N-hydroxysulphosuccinimide (sulphoNHS) were added to 2 mM and 5 mM respectively and the mixture incubated for 15 min at 25° C. The microparticles were washed by centrifugation for 1 min at 10000×g and resuspension in 1 ml of activation buffer. The wash step was repeated 4 times and then the microparticles resuspended in 1 ml of activation buffer containing 33 μM of a modified clostridial heavy chain fragment and incubated for 2 h at 25° C. The reaction was then quenched with 10 mM hydroxylamine. After 20 min at 25° C. the microparticles were washed in a suitable buffer by centrifugation as described above.

EXAMPLE 7

[0137] Demonstration of the High Affinity Binding to Neuronal Cell Tissue Displayed by Modified Heavy Chain Fragments

[0138] Clostridial neurotoxins may be labelled with 125-iodine using chloramine-T and its binding to various cells assessed by standard methods such as described in Evans et al. 1986, Eur J. Biochem., 154, 409 or Wadsworth et al. 1990, Biochem. J. 268, 123). In these experiments the ability of modified clostridial heavy chain constructs to compete with native clostridial neurotoxins for receptors present on neuronal cells or brain synaptosomes was assessed. All binding experiments were carried out in binding buffers. For the botulinum neurotoxins this buffer consisted of: 50 mM hepes pH 7.0, 30 mM NaCl, 0.25% sucrose, 0.25% bovine serum albumin. For tetanus toxin, the binding buffer was: 0.05M MES buffer pH 6.0 containing 0.6% bovine serum albumin. In a typical binding experiment the radiolabelled clostridial neurotoxin was held at a fixed concentration of between 1-10 nM. Reaction mixtures were prepared by mixing the radiolabelled toxin with various concentrations of unlabelled neurotoxin or modified clostridial heavy chain construct. The reaction mixture were then added to neuronal cells or rat brain synaptosomes and then incubated at 0-3° C. for 2 hr. After this period the neuronal cells of synaptosomes were washed twice with binding ice-cold binding buffer and the amount of labelled clostridial neurotoxin bound to cells or synaptosomes was assessed by γ-counting.

[0139] In an experiment using a modified clostridial heavy construct which consisted of a binding domain derived from tetanus toxin and a translocation domain from diphtheria toxin, the construct was found to compete with ¹²⁵I-labelled tetanus neurotoxin for neuronal cell receptors in a similar manner to unlabelled native tetanus neurotoxin (see FIG. 6). These data showed that the construct had retained binding properties of the native neurotoxin.

[0140] In a further experiment using Diphtheria H_(N)-BoNT/F H_(c) as the modified clostridial heavy chain, the construct was found to compete with 125I-labelled BoNT/F for receptors on neuronal synaptic membranes (FIG. 7). These data indicate that the modified clostridial heavy chain retains the neuronal receptor-binding properties of BoNT/F.

EXAMPLE 8

[0141] Non-Denaturing Gel Electrophoresis to Compare the Sizes of a Native Botulinum Toxin Heavy Chain (Type A) with that of a Modified Clostridial Heavy Chain (Recombinant Diphtheria H_(N)-BoNT/F H_(C))

[0142] Botulinum type A heavy chain was purified as described previously (Shone et al. 1985 Eur J. Biochemistry 151, 75-82) and recombinant Diphtheria H_(N)-BoNT/F H_(C) purified as described in Examples 1 and 2. The modified clostridial heavy chain was purifies as a Maltose Binding Protein fusion with then the fusion protein removed by treatment with Factor Xa. Samples of type A heavy chain (20 μg) and Diphtheria H_(N)-BoNT/F H_(C) (10 μg) were loaded on a 4-20% Tris-glycine polyacrylamide gel in Tris-glycine buffer. Samples were electrophoresed to equilibrium (Novex gel system; 43 volts 16 hours) and the gel stained with Coomassie blue. The results are shown in FIG. 8. The major band for Diphtheria H_(N)-BoNT/F/H_(C) appears to migrate very close to its predicted molecular weight of approx 70 kDa. In contrast, the native type A heavy chain appears as a diffuse band at approximately 500 kDa, compared to an estimated molecular weight of 100 kDa, which suggesting the formation of large protein aggregates.

EXAMPLE 9

[0143] Recombinant Modified Heavy Chain-Superoxide Dismutase Conjugates.

[0144] Recombinant modified heavy chain-superoxide dismutase conjugates were prepared comprising a combination of the following elements:

[0145] a bacterial superoxide dismutase, from Bacillus stearothermophilus;

[0146] a linker region which allows the formation of a disulphide bond between the superoxide dismutase and the translocation domain and which also contains a unique protease cleavage site for cleavage by factor Xa or thrombin to allow the formation of a dichain molecule;

[0147] a translocation domain from diphtheria toxin or a endosomolytic (fusogenic) peptide from influenza virus haemagglutinin); and

[0148] a neuronal cell-specific binding domain from tetanus or botulinum neurotoxin type F.

[0149] The sequences of these recombinant modified heavy chain-superoxide dismutase conjugates are shown in SEQ ID NO:s 3-7.

[0150] To confirm the nature of their structure, the recombinant modified clostridial heavy chain-superoxide dismutase conjugates were converted to the dichain form by treatment with a unique protease corresponding to the cleavage site sequences within the linker region. Conjugates containing the thrombin cleavage site were treated with thrombin (20 μg per mg of conjugate) for 20 h at 37° C.; conjugates containing the factor Xa cleavage site were treated with factor Xa (20 μg per mg of conjugate) for 20 min at 22° C.

[0151] On SDS-PAGE gels, under non-reducing conditions, the conjugates appeared as a band of molecular mass approx. 120 kDa. In the presence of reducing agent (dithiothreitol) two bands were observed at approx. molecular masses 70 and 30 kDa corresponding to the modified clostridial heavy chain and superoxide dismutase respectively. These data illustrate that, after treatment with the unique protease, the conjugates consist of the latter two components which are linked by a disulphide bridge. TABLE 1 Examples of Potential Therapeutic Uses of Modified Clostridial Binding Fragments Therapeutic Substance or Site and Mechanism Potential Effector of Action Clinical Effects (a) Enzymes:- Superoxide dismutase Reduce oxidative stress Reduction of after stroke/injury of brain neuronal damage or spinal cord after ischemia/ reperfusion Glutamine synthetase Reduce damage by excess Reduction of glutamate after stroke/injury neuronal damage of the brain or spinal cord after ischemia/ reperfusion (b) Antibodies:- Anti-tetanus toxin Neutralize the action of Reverse the effects tetanus toxin at the spinal of intoxication cord by tetanus toxin Anti SNARE protein Modulate neurotransmitter Hyper secretory (e.g. SNAP-25, VAMPs release disorders Syntaxins) © Viruses/DNA Viral gene Replacement of defective Treatment of therapy vectors genes within the CNS neurodegenerative (e.g. adenovirus, diseases (Parkinson's' herpes simplex, etc.) Alzheimer's ALS etc.) and other neuronal diseases Non-viral vectors Replacement of defective Treatment of for gene therapy genes within the CNS neurodegenerative (e.g. liposomes) diseases and other neuronal diseases (d) Growth factors e.g. BDNF, CTNF, NGF Deliver growth factors to Treatment of the brain and spinal cord neurodegenerative diseases, promotion of neuronal growth after damage (e) Anti-viral agents Deliver anti-viral agents Treatment of latent to the brain or spinal cord viral infections neurons within neuronal cells, e.g. HIV, herpes simplex infections (f) Anti-cancer agents Deliver cytotoxic agents Treatment of neuronal to neoplastic cells of the CNS neoplasia

[0152]

1 18 1 10 PRT Artificial Sequence synthetic construct 1 Cys Gly Leu Val Pro Ala Gly Ser Gly Pro 1 5 10 2 17 PRT Artificial Sequence synthetic construct 2 Cys Gly Leu Val Pro Ala Gly Ser Gly Pro Ser Ala Gly Ser Ser Ala 1 5 10 15 Cys 3 882 PRT Artificial Sequence synthetic construct 3 Met Pro Phe Glu Leu Pro Ala Leu Pro Tyr Pro Tyr Asp Ala Leu Glu 1 5 10 15 Pro His Ile Asp Lys Glu Thr Met Asn Ile His His Thr Lys His His 20 25 30 Asn Thr Tyr Val Thr Asn Leu Asn Ala Ala Leu Glu Gly His Pro Asp 35 40 45 Leu Gln Asn Lys Ser Leu Glu Glu Leu Leu Ser Asn Leu Glu Ala Leu 50 55 60 Pro Glu Ser Ile Arg Thr Ala Val Arg Asn Asn Gly Gly Gly His Ala 65 70 75 80 Asn His Ser Leu Phe Trp Thr Ile Leu Ser Pro Asn Gly Gly Gly Glu 85 90 95 Pro Thr Gly Glu Leu Ala Asp Ala Ile Asn Lys Lys Phe Gly Ser Phe 100 105 110 Thr Ala Phe Lys Asp Glu Phe Ser Lys Ala Ala Ala Gly Arg Phe Gly 115 120 125 Ser Gly Trp Ala Trp Leu Val Val Asn Asn Gly Glu Leu Glu Ile Thr 130 135 140 Ser Thr Pro Asn Gln Asp Ser Pro Ile Met Glu Gly Lys Thr Pro Ile 145 150 155 160 Leu Gly Leu Asp Val Trp Glu His Ala Tyr Tyr Leu Lys Tyr Gln Asn 165 170 175 Arg Arg Pro Glu Tyr Ile Ala Ala Phe Trp Asn Val Val Asn Trp Asp 180 185 190 Glu Val Ala Lys Arg Tyr Ser Glu Ala Lys Pro Lys Ser Gly Ser Cys 195 200 205 Gly Leu Val Pro Arg Gly Ser Gly Pro Gly Ser Ser Val Gly Ser Ser 210 215 220 Leu Ser Cys Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys Thr Lys 225 230 235 240 Thr Lys Ile Glu Ser Leu Lys Glu His Gly Pro Ile Lys Asn Lys Met 245 250 255 Ser Glu Ser Pro Asn Lys Thr Val Ser Glu Glu Lys Ala Lys Gln Tyr 260 265 270 Leu Glu Glu Phe His Gln Thr Ala Leu Glu His Pro Glu Leu Ser Glu 275 280 285 Leu Lys Thr Val Thr Gly Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr 290 295 300 Ala Ala Trp Ala Val Asn Val Ala Gln Val Ile Asp Ser Glu Thr Ala 305 310 315 320 Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile 325 330 335 Gly Ser Val Met Gly Ile Ala Asp Gly Ala Val His His Asn Thr Glu 340 345 350 Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala Gln 355 360 365 Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr 370 375 380 Asn Phe Val Glu Ser Ile Ile Asn Leu Phe Gln Val Val His Asn Ser 385 390 395 400 Tyr Asn Arg Ser Ala Tyr Ser Pro Gly His Lys Thr Gln Pro Phe Leu 405 410 415 His Asp Gly Tyr Ala Val Ser Trp Asn Thr Val Arg Ser Lys Asn Leu 420 425 430 Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu Lys Lys 435 440 445 Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile 450 455 460 Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln Leu Val 465 470 475 480 Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu Ser Ser 485 490 495 Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp Met Phe 500 505 510 Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala 515 520 525 Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser 530 535 540 Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val Ser Leu 545 550 555 560 Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu Val 565 570 575 Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu 580 585 590 Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser 595 600 605 Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala Glu Ile Thr 610 615 620 Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp 625 630 635 640 Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile 645 650 655 Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser 660 665 670 Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg 675 680 685 Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp 690 695 700 Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro 705 710 715 720 Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn 725 730 735 Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp 740 745 750 Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn 755 760 765 Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn 770 775 780 Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro 785 790 795 800 Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr 805 810 815 Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu 820 825 830 Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile 835 840 845 Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu 850 855 860 Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp 865 870 875 880 Leu Gln 4 862 PRT Artificial Sequence synthetic construct 4 Met Pro Phe Glu Leu Pro Ala Leu Pro Tyr Pro Tyr Asp Ala Leu Glu 1 5 10 15 Pro His Ile Asp Lys Glu Thr Met Asn Ile His His Thr Lys His His 20 25 30 Asn Thr Tyr Val Thr Asn Leu Asn Ala Ala Leu Glu Gly His Pro Asp 35 40 45 Leu Gln Asn Lys Ser Leu Glu Glu Leu Leu Ser Asn Leu Glu Ala Leu 50 55 60 Pro Glu Ser Ile Arg Thr Ala Val Arg Asn Asn Gly Gly Gly His Ala 65 70 75 80 Asn His Ser Leu Phe Trp Thr Ile Leu Ser Pro Asn Gly Gly Gly Glu 85 90 95 Pro Thr Gly Glu Leu Ala Asp Ala Ile Asn Lys Lys Phe Gly Ser Phe 100 105 110 Thr Ala Phe Lys Asp Glu Phe Ser Lys Ala Ala Ala Gly Arg Phe Gly 115 120 125 Ser Gly Trp Ala Trp Leu Val Val Asn Asn Gly Glu Leu Glu Ile Thr 130 135 140 Ser Thr Pro Asn Gln Asp Ser Pro Ile Met Glu Gly Lys Thr Pro Ile 145 150 155 160 Leu Gly Leu Asp Val Trp Glu His Ala Tyr Tyr Leu Lys Tyr Gln Asn 165 170 175 Arg Arg Pro Glu Tyr Ile Ala Ala Phe Trp Asn Val Val Asn Trp Asp 180 185 190 Glu Val Ala Lys Arg Tyr Ser Glu Ala Lys Pro Lys Ser Gly Ser Cys 195 200 205 Gly Ile Glu Gly Arg Ala Pro Gly Pro Gly Ser Ser Val Gly Ser Ser 210 215 220 Leu Ser Cys Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys Thr Lys 225 230 235 240 Thr Lys Ile Glu Ser Leu Lys Glu His Gly Pro Ile Lys Asn Lys Met 245 250 255 Ser Glu Ser Pro Asn Lys Thr Val Ser Glu Glu Lys Ala Lys Gln Tyr 260 265 270 Leu Glu Glu Phe His Gln Thr Ala Leu Glu His Pro Glu Leu Ser Glu 275 280 285 Leu Lys Thr Val Thr Gly Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr 290 295 300 Ala Ala Trp Ala Val Asn Val Ala Gln Val Ile Asp Ser Glu Thr Ala 305 310 315 320 Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile 325 330 335 Gly Ser Val Met Gly Ile Ala Asp Gly Ala Val His His Asn Thr Glu 340 345 350 Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala Gln 355 360 365 Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr 370 375 380 Asn Phe Val Glu Ser Ile Ile Asn Leu Phe Gln Val Val His Asn Ser 385 390 395 400 Tyr Asn Arg Ser Ala Tyr Ser Pro Gly His Lys Thr Gln Pro Phe Leu 405 410 415 His Asp Gly Tyr Ala Val Ser Trp Asn Thr Val Arg Ser Thr Met Ser 420 425 430 Tyr Thr Asn Asp Lys Ile Leu Ile Leu Tyr Phe Asn Lys Leu Tyr Lys 435 440 445 Lys Ile Lys Asp Asn Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys 450 455 460 Phe Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asp 465 470 475 480 Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Ser Ser 485 490 495 Lys Pro Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn 500 505 510 Gly Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys 515 520 525 Tyr Phe Asn Lys Val Asn Leu Asn Asn Glu Tyr Thr Ile Ile Asp Cys 530 535 540 Ile Arg Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Asn Tyr Asn Lys 545 550 555 560 Ile Ile Trp Thr Leu Gln Asp Thr Ala Gly Asn Asn Gln Lys Leu Val 565 570 575 Phe Asn Tyr Thr Gln Met Ile Ser Ile Ser Asp Tyr Ile Asn Lys Trp 580 585 590 Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg Ile Tyr 595 600 605 Ile Asn Gly Asn Leu Ile Asp Glu Lys Ser Ile Ser Asn Leu Gly Asp 610 615 620 Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys Asn Asp 625 630 635 640 Thr Arg Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asp Thr Glu Leu 645 650 655 Gly Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asp Glu Pro Asp Pro Ser 660 665 670 Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asn Lys Arg Tyr 675 680 685 Tyr Leu Leu Asn Leu Leu Arg Thr Asp Lys Ser Ile Thr Gln Asn Ser 690 695 700 Asn Phe Leu Asn Ile Asn Gln Gln Arg Gly Val Tyr Gln Lys Pro Asn 705 710 715 720 Ile Phe Ser Asn Thr Arg Leu Tyr Thr Gly Val Glu Val Ile Ile Arg 725 730 735 Lys Asn Gly Ser Thr Asp Ile Ser Asn Thr Asp Asn Phe Val Arg Lys 740 745 750 Asn Asp Leu Ala Tyr Ile Asn Val Val Asp Arg Asp Val Glu Tyr Arg 755 760 765 Leu Tyr Ala Asp Ile Ser Ile Ala Lys Pro Glu Lys Ile Ile Lys Leu 770 775 780 Ile Arg Thr Ser Asn Ser Asn Asn Ser Leu Gly Gln Ile Ile Val Met 785 790 795 800 Asp Ser Ile Gly Asn Asn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly 805 810 815 Gly Asn Ile Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser 820 825 830 Ser Trp Tyr Tyr Asn Asn Ile Arg Lys Asn Thr Ser Ser Asn Gly Cys 835 840 845 Phe Trp Ser Phe Ile Ser Lys Glu His Gly Trp Gln Glu Asn 850 855 860 5 907 PRT Artificial Sequence synthetic construct 5 Met Leu Ser Arg Ala Val Cys Gly Thr Ser Arg Gln Leu Ala Pro Ala 1 5 10 15 Leu Gly Tyr Leu Gly Ser Arg Gln Lys His Ser Arg Gly Ser Pro Ala 20 25 30 Leu Pro Tyr Pro Tyr Asp Ala Leu Glu Pro His Ile Asp Lys Glu Thr 35 40 45 Met Asn Ile His His Thr Lys His His Asn Thr Tyr Val Thr Asn Leu 50 55 60 Asn Ala Ala Leu Glu Gly His Pro Asp Leu Gln Asn Lys Ser Leu Glu 65 70 75 80 Glu Leu Leu Ser Asn Leu Glu Ala Leu Pro Glu Ser Ile Arg Thr Ala 85 90 95 Val Arg Asn Asn Gly Gly Gly His Ala Asn His Ser Leu Phe Trp Thr 100 105 110 Ile Leu Ser Pro Asn Gly Gly Gly Glu Pro Thr Gly Glu Leu Ala Asp 115 120 125 Ala Ile Asn Lys Lys Phe Gly Ser Phe Thr Ala Phe Lys Asp Glu Phe 130 135 140 Ser Lys Ala Ala Ala Gly Arg Phe Gly Ser Gly Trp Ala Trp Leu Val 145 150 155 160 Val Asn Asn Gly Glu Leu Glu Ile Thr Ser Thr Pro Asn Gln Asp Ser 165 170 175 Pro Ile Met Glu Gly Lys Thr Pro Ile Leu Gly Leu Asp Val Trp Glu 180 185 190 His Ala Tyr Tyr Leu Lys Tyr Gln Asn Arg Arg Pro Glu Tyr Ile Ala 195 200 205 Ala Phe Trp Asn Val Val Asn Trp Asp Glu Val Ala Lys Arg Tyr Ser 210 215 220 Glu Ala Lys Pro Lys Ser Gly Ser Cys Gly Ile Glu Gly Arg Ala Pro 225 230 235 240 Gly Pro Gly Ser Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp 245 250 255 Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys 260 265 270 Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr 275 280 285 Val Ser Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr 290 295 300 Ala Leu Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr 305 310 315 320 Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val 325 330 335 Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr 340 345 350 Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala 355 360 365 Asp Gly Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile 370 375 380 Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu 385 390 395 400 Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile 405 410 415 Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Ser Ala Tyr Ser 420 425 430 Pro Gly His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser 435 440 445 Trp Asn Thr Val Arg Ser Lys Asn Leu Asp Cys Trp Val Asp Asn Glu 450 455 460 Glu Asp Ile Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp 465 470 475 480 Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser Val 485 490 495 Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys Ala 500 505 510 Ile His Leu Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys Ala 515 520 525 Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser Phe 530 535 540 Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr Gly 545 550 555 560 Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser 565 570 575 Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp 580 585 590 Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp 595 600 605 Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile 610 615 620 Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly 625 630 635 640 Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu 645 650 655 Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln 660 665 670 Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro 675 680 685 Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu 690 695 700 Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu 705 710 715 720 Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile Thr 725 730 735 Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu 740 745 750 Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys 755 760 765 Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly Asp 770 775 780 Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val Gly 785 790 795 800 Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg 805 810 815 Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala 820 825 830 Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr 835 840 845 Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln 850 855 860 Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr 865 870 875 880 Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val 885 890 895 Pro Thr Asp Glu Gly Trp Thr Asn Asp Leu Gln 900 905 6 887 PRT Artificial Sequence synthetic construct 6 Met Leu Ser Arg Ala Val Cys Gly Thr Ser Arg Gln Leu Ala Pro Ala 1 5 10 15 Leu Gly Tyr Leu Gly Ser Arg Gln Lys His Ser Arg Gly Ser Pro Ala 20 25 30 Leu Pro Tyr Pro Tyr Asp Ala Leu Glu Pro His Ile Asp Lys Glu Thr 35 40 45 Met Asn Ile His His Thr Lys His His Asn Thr Tyr Val Thr Asn Leu 50 55 60 Asn Ala Ala Leu Glu Gly His Pro Asp Leu Gln Asn Lys Ser Leu Glu 65 70 75 80 Glu Leu Leu Ser Asn Leu Glu Ala Leu Pro Glu Ser Ile Arg Thr Ala 85 90 95 Val Arg Asn Asn Gly Gly Gly His Ala Asn His Ser Leu Phe Trp Thr 100 105 110 Ile Leu Ser Pro Asn Gly Gly Gly Glu Pro Thr Gly Glu Leu Ala Asp 115 120 125 Ala Ile Asn Lys Lys Phe Gly Ser Phe Thr Ala Phe Lys Asp Glu Phe 130 135 140 Ser Lys Ala Ala Ala Gly Arg Phe Gly Ser Gly Trp Ala Trp Leu Val 145 150 155 160 Val Asn Asn Gly Glu Leu Glu Ile Thr Ser Thr Pro Asn Gln Asp Ser 165 170 175 Pro Ile Met Glu Gly Lys Thr Pro Ile Leu Gly Leu Asp Val Trp Glu 180 185 190 His Ala Tyr Tyr Leu Lys Tyr Gln Asn Arg Arg Pro Glu Tyr Ile Ala 195 200 205 Ala Phe Trp Asn Val Val Asn Trp Asp Glu Val Ala Lys Arg Tyr Ser 210 215 220 Glu Ala Lys Pro Lys Ser Gly Ser Cys Gly Leu Val Pro Arg Gly Ser 225 230 235 240 Gly Pro Gly Ser Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp 245 250 255 Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys 260 265 270 Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr 275 280 285 Val Ser Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr 290 295 300 Ala Leu Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr 305 310 315 320 Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val 325 330 335 Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr 340 345 350 Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala 355 360 365 Asp Gly Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile 370 375 380 Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu 385 390 395 400 Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile 405 410 415 Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Ser Ala Tyr Ser 420 425 430 Pro Gly His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser 435 440 445 Trp Asn Thr Val Arg Ser Thr Met Ser Tyr Thr Asn Asp Lys Ile Leu 450 455 460 Ile Leu Tyr Phe Asn Lys Leu Tyr Lys Lys Ile Lys Asp Asn Ser Ile 465 470 475 480 Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe Ile Asp Ile Ser Gly Tyr 485 490 495 Gly Ser Asn Ile Ser Ile Asn Gly Asp Val Tyr Ile Tyr Ser Thr Asn 500 505 510 Arg Asn Gln Phe Gly Ile Tyr Ser Ser Lys Pro Ser Glu Val Asn Ile 515 520 525 Ala Gln Asn Asn Asp Ile Ile Tyr Asn Gly Arg Tyr Gln Asn Phe Ser 530 535 540 Ile Ser Phe Trp Val Arg Ile Pro Lys Tyr Phe Asn Lys Val Asn Leu 545 550 555 560 Asn Asn Glu Tyr Thr Ile Ile Asp Cys Ile Arg Asn Asn Asn Ser Gly 565 570 575 Trp Lys Ile Ser Leu Asn Tyr Asn Lys Ile Ile Trp Thr Leu Gln Asp 580 585 590 Thr Ala Gly Asn Asn Gln Lys Leu Val Phe Asn Tyr Thr Gln Met Ile 595 600 605 Ser Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile Thr Asn 610 615 620 Asn Arg Leu Gly Asn Ser Arg Ile Tyr Ile Asn Gly Asn Leu Ile Asp 625 630 635 640 Glu Lys Ser Ile Ser Asn Leu Gly Asp Ile His Val Ser Asp Asn Ile 645 650 655 Leu Phe Lys Ile Val Gly Cys Asn Asp Thr Arg Tyr Val Gly Ile Arg 660 665 670 Tyr Phe Lys Val Phe Asp Thr Glu Leu Gly Lys Thr Glu Ile Glu Thr 675 680 685 Leu Tyr Ser Asp Glu Pro Asp Pro Ser Ile Leu Lys Asp Phe Trp Gly 690 695 700 Asn Tyr Leu Leu Tyr Asn Lys Arg Tyr Tyr Leu Leu Asn Leu Leu Arg 705 710 715 720 Thr Asp Lys Ser Ile Thr Gln Asn Ser Asn Phe Leu Asn Ile Asn Gln 725 730 735 Gln Arg Gly Val Tyr Gln Lys Pro Asn Ile Phe Ser Asn Thr Arg Leu 740 745 750 Tyr Thr Gly Val Glu Val Ile Ile Arg Lys Asn Gly Ser Thr Asp Ile 755 760 765 Ser Asn Thr Asp Asn Phe Val Arg Lys Asn Asp Leu Ala Tyr Ile Asn 770 775 780 Val Val Asp Arg Asp Val Glu Tyr Arg Leu Tyr Ala Asp Ile Ser Ile 785 790 795 800 Ala Lys Pro Glu Lys Ile Ile Lys Leu Ile Arg Thr Ser Asn Ser Asn 805 810 815 Asn Ser Leu Gly Gln Ile Ile Val Met Asp Ser Ile Gly Asn Asn Cys 820 825 830 Thr Met Asn Phe Gln Asn Asn Asn Gly Gly Asn Ile Gly Leu Leu Gly 835 840 845 Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr Tyr Asn Asn Ile 850 855 860 Arg Lys Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser Phe Ile Ser Lys 865 870 875 880 Glu His Gly Trp Gln Glu Asn 885 7 685 PRT Artificial Sequence synthetic construct 7 Met Pro Phe Glu Leu Pro Ala Leu Pro Tyr Pro Tyr Asp Ala Leu Glu 1 5 10 15 Pro His Ile Asp Lys Glu Thr Met Asn Ile His His Thr Lys His His 20 25 30 Asn Thr Tyr Val Thr Asn Leu Asn Ala Ala Leu Glu Gly His Pro Asp 35 40 45 Leu Gln Asn Lys Ser Leu Glu Glu Leu Leu Ser Asn Leu Glu Ala Leu 50 55 60 Pro Glu Ser Ile Arg Thr Ala Val Arg Asn Asn Gly Gly Gly His Ala 65 70 75 80 Asn His Ser Leu Phe Trp Thr Ile Leu Ser Pro Asn Gly Gly Gly Glu 85 90 95 Pro Thr Gly Glu Leu Ala Asp Ala Ile Asn Lys Lys Phe Gly Ser Phe 100 105 110 Thr Ala Phe Lys Asp Glu Phe Ser Lys Ala Ala Ala Gly Arg Phe Gly 115 120 125 Ser Gly Trp Ala Trp Leu Val Val Asn Asn Gly Glu Leu Glu Ile Thr 130 135 140 Ser Thr Pro Asn Gln Asp Ser Pro Ile Met Glu Gly Lys Thr Pro Ile 145 150 155 160 Leu Gly Leu Asp Val Trp Glu His Ala Tyr Tyr Leu Lys Tyr Gln Asn 165 170 175 Arg Arg Pro Glu Tyr Ile Ala Ala Phe Trp Asn Val Val Asn Trp Asp 180 185 190 Glu Val Ala Lys Arg Tyr Ser Glu Ala Lys Pro Lys Ser Gly Ser Cys 195 200 205 Gly Ile Glu Gly Arg Ala Pro Gly Pro Gly Ser Ser Val Gly Ser Ser 210 215 220 Leu Ser Cys Ile Asn Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 225 230 235 240 Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Thr Met Ser Tyr 245 250 255 Thr Asn Asp Lys Ile Leu Ile Leu Tyr Phe Asn Lys Leu Tyr Lys Lys 260 265 270 Ile Lys Asp Asn Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe 275 280 285 Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asp Val 290 295 300 Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Ser Ser Lys 305 310 315 320 Pro Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn Gly 325 330 335 Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys Tyr 340 345 350 Phe Asn Lys Val Asn Leu Asn Asn Glu Tyr Thr Ile Ile Asp Cys Ile 355 360 365 Arg Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Asn Tyr Asn Lys Ile 370 375 380 Ile Trp Thr Leu Gln Asp Thr Ala Gly Asn Asn Gln Lys Leu Val Phe 385 390 395 400 Asn Tyr Thr Gln Met Ile Ser Ile Ser Asp Tyr Ile Asn Lys Trp Ile 405 410 415 Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg Ile Tyr Ile 420 425 430 Asn Gly Asn Leu Ile Asp Glu Lys Ser Ile Ser Asn Leu Gly Asp Ile 435 440 445 His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys Asn Asp Thr 450 455 460 Arg Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asp Thr Glu Leu Gly 465 470 475 480 Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asp Glu Pro Asp Pro Ser Ile 485 490 495 Leu Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asn Lys Arg Tyr Tyr 500 505 510 Leu Leu Asn Leu Leu Arg Thr Asp Lys Ser Ile Thr Gln Asn Ser Asn 515 520 525 Phe Leu Asn Ile Asn Gln Gln Arg Gly Val Tyr Gln Lys Pro Asn Ile 530 535 540 Phe Ser Asn Thr Arg Leu Tyr Thr Gly Val Glu Val Ile Ile Arg Lys 545 550 555 560 Asn Gly Ser Thr Asp Ile Ser Asn Thr Asp Asn Phe Val Arg Lys Asn 565 570 575 Asp Leu Ala Tyr Ile Asn Val Val Asp Arg Asp Val Glu Tyr Arg Leu 580 585 590 Tyr Ala Asp Ile Ser Ile Ala Lys Pro Glu Lys Ile Ile Lys Leu Ile 595 600 605 Arg Thr Ser Asn Ser Asn Asn Ser Leu Gly Gln Ile Ile Val Met Asp 610 615 620 Ser Ile Gly Asn Asn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly Gly 625 630 635 640 Asn Ile Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser 645 650 655 Trp Tyr Tyr Asn Asn Ile Arg Lys Asn Thr Ser Ser Asn Gly Cys Phe 660 665 670 Trp Ser Phe Ile Ser Lys Glu His Gly Trp Gln Glu Asn 675 680 685 8 645 PRT Artificial Sequence synthetic construct 8 Gly Ser Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp 1 5 10 15 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His 20 25 30 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser 35 40 45 Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 50 55 60 Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 65 70 75 80 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 85 90 95 Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 100 105 110 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly 115 120 125 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu 130 135 140 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val 145 150 155 160 Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu 165 170 175 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 180 185 190 His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn 195 200 205 Thr Val Arg Ser Thr Met Ser Tyr Thr Asn Asp Lys Ile Leu Ile Leu 210 215 220 Tyr Phe Asn Lys Leu Tyr Lys Lys Ile Lys Asp Asn Ser Ile Leu Asp 225 230 235 240 Met Arg Tyr Glu Asn Asn Lys Phe Ile Asp Ile Ser Gly Tyr Gly Ser 245 250 255 Asn Ile Ser Ile Asn Gly Asp Val Tyr Ile Tyr Ser Thr Asn Arg Asn 260 265 270 Gln Phe Gly Ile Tyr Ser Ser Lys Pro Ser Glu Val Asn Ile Ala Gln 275 280 285 Asn Asn Asp Ile Ile Tyr Asn Gly Arg Tyr Gln Asn Phe Ser Ile Ser 290 295 300 Phe Trp Val Arg Ile Pro Lys Tyr Phe Asn Lys Val Asn Leu Asn Asn 305 310 315 320 Glu Tyr Thr Ile Ile Asp Cys Ile Arg Asn Asn Asn Ser Gly Trp Lys 325 330 335 Ile Ser Leu Asn Tyr Asn Lys Ile Ile Trp Thr Leu Gln Asp Thr Ala 340 345 350 Gly Asn Asn Gln Lys Leu Val Phe Asn Tyr Thr Gln Met Ile Ser Ile 355 360 365 Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg 370 375 380 Leu Gly Asn Ser Arg Ile Tyr Ile Asn Gly Asn Leu Ile Asp Glu Lys 385 390 395 400 Ser Ile Ser Asn Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe 405 410 415 Lys Ile Val Gly Cys Asn Asp Thr Arg Tyr Val Gly Ile Arg Tyr Phe 420 425 430 Lys Val Phe Asp Thr Glu Leu Gly Lys Thr Glu Ile Glu Thr Leu Tyr 435 440 445 Ser Asp Glu Pro Asp Pro Ser Ile Leu Lys Asp Phe Trp Gly Asn Tyr 450 455 460 Leu Leu Tyr Asn Lys Arg Tyr Tyr Leu Leu Asn Leu Leu Arg Thr Asp 465 470 475 480 Lys Ser Ile Thr Gln Asn Ser Asn Phe Leu Asn Ile Asn Gln Gln Arg 485 490 495 Gly Val Tyr Gln Lys Pro Asn Ile Phe Ser Asn Thr Arg Leu Tyr Thr 500 505 510 Gly Val Glu Val Ile Ile Arg Lys Asn Gly Ser Thr Asp Ile Ser Asn 515 520 525 Thr Asp Asn Phe Val Arg Lys Asn Asp Leu Ala Tyr Ile Asn Val Val 530 535 540 Asp Arg Asp Val Glu Tyr Arg Leu Tyr Ala Asp Ile Ser Ile Ala Lys 545 550 555 560 Pro Glu Lys Ile Ile Lys Leu Ile Arg Thr Ser Asn Ser Asn Asn Ser 565 570 575 Leu Gly Gln Ile Ile Val Met Asp Ser Ile Gly Asn Asn Cys Thr Met 580 585 590 Asn Phe Gln Asn Asn Asn Gly Gly Asn Ile Gly Leu Leu Gly Phe His 595 600 605 Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr Tyr Asn Asn Ile Arg Lys 610 615 620 Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser Phe Ile Ser Lys Glu His 625 630 635 640 Gly Trp Gln Glu Asn 645 9 665 PRT Artificial Sequence synthetic construct 9 Gly Ser Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp 1 5 10 15 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His 20 25 30 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser 35 40 45 Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 50 55 60 Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 65 70 75 80 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 85 90 95 Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 100 105 110 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly 115 120 125 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu 130 135 140 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val 145 150 155 160 Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu 165 170 175 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 180 185 190 His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn 195 200 205 Thr Val Arg Ser Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp 210 215 220 Ile Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn 225 230 235 240 Asn Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr 245 250 255 Tyr Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His 260 265 270 Leu Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp 275 280 285 Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu 290 295 300 Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn 305 310 315 320 Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly 325 330 335 Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu 340 345 350 Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro 355 360 365 Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile 370 375 380 Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu 385 390 395 400 Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn 405 410 415 Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val 420 425 430 Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu 435 440 445 Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp 450 455 460 Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro 465 470 475 480 Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr 485 490 495 Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile 500 505 510 Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr 515 520 525 Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile 530 535 540 Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro 545 550 555 560 Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly 565 570 575 Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys 580 585 590 Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp 595 600 605 Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly 610 615 620 Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn 625 630 635 640 His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr 645 650 655 Asp Glu Gly Trp Thr Asn Asp Leu Gln 660 665 10 448 PRT Artificial Sequence synthetic construct 10 Gly Ser Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp 1 5 10 15 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His 20 25 30 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser 35 40 45 Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 50 55 60 Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 65 70 75 80 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 85 90 95 Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 100 105 110 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly 115 120 125 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu 130 135 140 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val 145 150 155 160 Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu 165 170 175 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 180 185 190 His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn 195 200 205 Thr Val Arg Ser Val Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys 210 215 220 Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu 225 230 235 240 Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp 245 250 255 Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln 260 265 270 Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr 275 280 285 Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu 290 295 300 Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe 305 310 315 320 Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn 325 330 335 Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu 340 345 350 Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr 355 360 365 Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val 370 375 380 Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly 385 390 395 400 Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile 405 410 415 Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys 420 425 430 Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp Leu Gln 435 440 445 11 605 PRT Artificial Sequence synthetic construct 11 Gly Ser Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp 1 5 10 15 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His 20 25 30 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser 35 40 45 Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 50 55 60 Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 65 70 75 80 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 85 90 95 Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 100 105 110 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly 115 120 125 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu 130 135 140 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val 145 150 155 160 Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu 165 170 175 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 180 185 190 His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn 195 200 205 Thr Val Arg Ser Val Tyr Asn Asn Glu Ser Ser Glu Val Ile Val His 210 215 220 Lys Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val 225 230 235 240 Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Gln 245 250 255 Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser 260 265 270 Leu Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu 275 280 285 Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe 290 295 300 Arg Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val 305 310 315 320 Phe Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile 325 330 335 Asn Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile 340 345 350 Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn 355 360 365 Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu 370 375 380 Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr 385 390 395 400 Phe Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr 405 410 415 Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn 420 425 430 Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly 435 440 445 Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile 450 455 460 Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser 465 470 475 480 Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile 485 490 495 Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile 500 505 510 Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met 515 520 525 Glu Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys 530 535 540 Leu Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn 545 550 555 560 Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn 565 570 575 Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr 580 585 590 Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp Leu Gln 595 600 605 12 660 PRT Artificial Sequence synthetic construct 12 Gly Ser Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp 1 5 10 15 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His 20 25 30 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser 35 40 45 Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu 50 55 60 Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 65 70 75 80 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 85 90 95 Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 100 105 110 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly 115 120 125 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu 130 135 140 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val 145 150 155 160 Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu 165 170 175 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 180 185 190 His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn 195 200 205 Thr Val Arg Ser Thr Met Ser Tyr Thr Asn Asp Lys Ile Leu Ile Leu 210 215 220 Tyr Phe Asn Lys Leu Tyr Lys Lys Ile Lys Asp Asn Ser Ile Leu Asp 225 230 235 240 Met Arg Tyr Glu Asn Asn Lys Phe Ile Asp Ile Ser Gly Tyr Gly Ser 245 250 255 Asn Ile Ser Ile Asn Gly Asp Val Tyr Ile Tyr Ser Thr Asn Arg Asn 260 265 270 Gln Phe Gly Ile Tyr Ser Ser Lys Pro Ser Glu Val Asn Ile Ala Gln 275 280 285 Asn Asn Asp Ile Ile Tyr Asn Gly Arg Tyr Gln Asn Phe Ser Ile Ser 290 295 300 Phe Trp Val Arg Ile Pro Lys Tyr Phe Asn Lys Val Asn Leu Asn Asn 305 310 315 320 Glu Tyr Thr Ile Ile Asp Cys Ile Arg Asn Asn Asn Ser Gly Trp Lys 325 330 335 Ile Ser Leu Asn Tyr Asn Lys Ile Ile Trp Thr Leu Gln Asp Thr Ala 340 345 350 Gly Asn Asn Gln Lys Leu Val Phe Asn Tyr Thr Gln Met Ile Ser Ile 355 360 365 Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg 370 375 380 Leu Gly Asn Ser Arg Ile Tyr Ile Asn Gly Asn Leu Ile Asp Glu Lys 385 390 395 400 Ser Ile Ser Asn Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe 405 410 415 Lys Ile Val Gly Cys Asn Asp Thr Arg Tyr Val Ser Ile Asp Lys Phe 420 425 430 Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr 435 440 445 Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro 450 455 460 Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser 465 470 475 480 Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn 485 490 495 Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu 500 505 510 Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu 515 520 525 Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser 530 535 540 Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala 545 550 555 560 Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly 565 570 575 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys 580 585 590 Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu 595 600 605 Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg 610 615 620 Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp Lys 625 630 635 640 Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr 645 650 655 Asn Asp Leu Gln 660 13 210 PRT Corynebacterium diphtheriae 13 Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val Ile 1 5 10 15 Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly Pro 20 25 30 Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu Glu 35 40 45 Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu His 50 55 60 Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val Phe 65 70 75 80 Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val Ile 85 90 95 Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu Ser 100 105 110 Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala Val 115 120 125 His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser 130 135 140 Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile 145 150 155 160 Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe Gln 165 170 175 Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His Lys 180 185 190 Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr Val 195 200 205 Arg Ser 210 14 1092 PRT Clostridium botulinum 14 Leu Val Ser Lys Phe Glu Asn Ser Val Lys Asn Ser Asn Lys Asn Tyr 1 5 10 15 Phe Thr Ile Asn Gly Leu Met Gly Tyr Tyr Phe Glu Asn Asp Phe Phe 20 25 30 Asn Leu Asn Ile Ile Ser Pro Thr Leu Asp Gly Asn Leu Thr Phe Ser 35 40 45 Lys Glu Asp Ile Asn Ser Ile Leu Gly Asn Lys Ile Ile Lys Ser Ala 50 55 60 Arg Trp Ile Gly Leu Ile Lys Pro Ser Ile Thr Gly Glu Tyr Ile Leu 65 70 75 80 Ser Thr Asn Ser Pro Asn Cys Arg Val Glu Leu Asn Gly Glu Ile Phe 85 90 95 Asn Leu Ser Leu Asn Thr Ser Asn Thr Val Asn Leu Ile Gln Gly Asn 100 105 110 Val Tyr Asp Ile Arg Ile Glu Gln Leu Met Ser Glu Asn Gln Leu Leu 115 120 125 Lys Asn Tyr Glu Gly Ile Lys Leu Tyr Trp Glu Thr Ser Asp Ile Ile 130 135 140 Lys Glu Ile Ile Pro Ser Glu Val Leu Leu Lys Pro Asn Tyr Ser Asn 145 150 155 160 Thr Asn Glu Lys Ser Lys Phe Ile Pro Asn Asn Thr Leu Phe Ser Asn 165 170 175 Ala Lys Leu Lys Ala Asn Ala Asn Arg Asp Thr Asp Arg Asp Gly Ile 180 185 190 Pro Asp Glu Trp Glu Ile Asn Gly Tyr Thr Val Met Asn Gln Lys Ala 195 200 205 Val Ala Trp Asp Asp Lys Phe Ala Ala Asn Gly Tyr Lys Lys Tyr Val 210 215 220 Ser Asn Pro Phe Lys Pro Cys Thr Ala Asn Asp Pro Tyr Thr Asp Phe 225 230 235 240 Glu Lys Val Ser Gly Gln Ile Asp Pro Ser Val Ser Met Val Ala Arg 245 250 255 Asp Pro Met Ile Ser Ala Tyr Pro Ile Val Gly Val Gln Met Glu Arg 260 265 270 Leu Val Val Ser Lys Ser Glu Thr Ile Thr Gly Asp Ser Thr Lys Ser 275 280 285 Met Ser Lys Ser Thr Ser His Ser Ser Thr Asn Ile Asn Thr Val Gly 290 295 300 Ala Glu Val Ser Gly Ser Leu Gln Leu Ala Gly Gly Ile Phe Pro Val 305 310 315 320 Phe Ser Met Ser Ala Ser Ala Asn Tyr Ser His Thr Trp Gln Asn Thr 325 330 335 Ser Thr Val Asp Asp Thr Thr Gly Glu Ser Phe Ser Gln Gly Leu Ser 340 345 350 Ile Asn Thr Gly Glu Ser Ala Tyr Ile Asn Pro Asn Ile Arg Tyr Tyr 355 360 365 Asn Thr Gly Thr Ala Pro Val Tyr Asn Val Thr Pro Thr Thr Thr Ile 370 375 380 Val Ile Asp Lys Gln Ser Val Ala Thr Ile Lys Gly Gln Glu Ser Leu 385 390 395 400 Ile Gly Asp Tyr Leu Asn Pro Gly Gly Thr Tyr Pro Ile Ile Gly Glu 405 410 415 Pro Pro Met Ala Leu Asn Thr Met Asp Gln Phe Ser Ser Arg Leu Ile 420 425 430 Pro Ile Asn Tyr Asn Gln Leu Lys Ser Ile Asp Asn Gly Gly Thr Val 435 440 445 Met Leu Ser Thr Ser Gln Phe Thr Gly Asn Phe Ala Lys Tyr Asn Ser 450 455 460 Asn Gly Asn Leu Val Thr Asp Gly Asn Asn Trp Gly Pro Tyr Leu Gly 465 470 475 480 Thr Ile Lys Ser Thr Thr Ala Ser Leu Thr Leu Ser Phe Ser Gly Gln 485 490 495 Thr Thr Gln Val Ala Val Val Ala Pro Asn Phe Ser Asp Pro Glu Asp 500 505 510 Lys Thr Pro Lys Leu Thr Leu Glu Gln Ala Leu Val Lys Ala Phe Ala 515 520 525 Leu Glu Lys Lys Asn Gly Lys Phe Tyr Phe His Gly Leu Glu Ile Ser 530 535 540 Lys Asn Glu Lys Ile Gln Val Phe Leu Asp Ser Asn Thr Asn Asn Asp 545 550 555 560 Phe Glu Asn Gln Leu Lys Asn Thr Ala Asp Lys Asp Ile Met His Cys 565 570 575 Ile Ile Lys Arg Asn Met Asn Ile Leu Val Lys Val Ile Thr Phe Lys 580 585 590 Glu Asn Ile Ser Ser Ile Asn Ile Ile Asn Asp Thr Asn Phe Gly Val 595 600 605 Gln Ser Met Thr Gly Leu Ser Asn Arg Ser Lys Gly Gln Asp Gly Ile 610 615 620 Tyr Arg Ala Ala Thr Thr Ala Phe Ser Phe Lys Ser Lys Glu Leu Lys 625 630 635 640 Tyr Pro Glu Gly Arg Tyr Arg Met Arg Phe Val Ile Gln Ser Tyr Glu 645 650 655 Pro Phe Thr Thr Met Ser Tyr Thr Asn Asp Lys Ile Leu Ile Leu Tyr 660 665 670 Phe Asn Lys Leu Tyr Lys Lys Ile Lys Asp Asn Ser Ile Leu Asp Met 675 680 685 Arg Tyr Glu Asn Asn Lys Phe Ile Asp Ile Ser Gly Tyr Gly Ser Asn 690 695 700 Ile Ser Ile Asn Gly Asp Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gln 705 710 715 720 Phe Gly Ile Tyr Ser Ser Lys Pro Ser Glu Val Asn Ile Ala Gln Asn 725 730 735 Asn Asp Ile Ile Tyr Asn Gly Arg Tyr Gln Asn Phe Ser Ile Ser Phe 740 745 750 Trp Val Arg Ile Pro Lys Tyr Phe Asn Lys Val Asn Leu Asn Asn Glu 755 760 765 Tyr Thr Ile Ile Asp Cys Ile Arg Asn Asn Asn Ser Gly Trp Lys Ile 770 775 780 Ser Leu Asn Tyr Asn Lys Ile Ile Trp Thr Leu Gln Asp Thr Ala Gly 785 790 795 800 Asn Asn Gln Lys Leu Val Phe Asn Tyr Thr Gln Met Ile Ser Ile Ser 805 810 815 Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 820 825 830 Gly Asn Ser Arg Ile Tyr Ile Asn Gly Asn Leu Ile Asp Glu Lys Ser 835 840 845 Ile Ser Asn Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys 850 855 860 Ile Val Gly Cys Asn Asp Thr Arg Tyr Val Gly Ile Arg Tyr Phe Lys 865 870 875 880 Val Phe Asp Thr Glu Leu Gly Lys Thr Glu Ile Glu Thr Leu Tyr Ser 885 890 895 Asp Glu Pro Asp Pro Ser Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu 900 905 910 Leu Tyr Asn Lys Arg Tyr Tyr Leu Leu Asn Leu Leu Arg Thr Asp Lys 915 920 925 Ser Ile Thr Gln Asn Ser Asn Phe Leu Asn Ile Asn Gln Gln Arg Gly 930 935 940 Val Tyr Gln Lys Pro Asn Ile Phe Ser Asn Thr Arg Leu Tyr Thr Gly 945 950 955 960 Val Glu Val Ile Ile Arg Lys Asn Gly Ser Thr Asp Ile Ser Asn Thr 965 970 975 Asp Asn Phe Val Arg Lys Asn Asp Leu Ala Tyr Ile Asn Val Val Asp 980 985 990 Arg Asp Val Glu Tyr Arg Leu Tyr Ala Asp Ile Ser Ile Ala Lys Pro 995 1000 1005 Glu Lys Ile Ile Lys Leu Ile Arg Thr Ser Asn Ser Asn Asn Ser 1010 1015 1020 Leu Gly Gln Ile Ile Val Met Asp Ser Ile Gly Asn Asn Cys Thr 1025 1030 1035 Met Asn Phe Gln Asn Asn Asn Gly Gly Asn Ile Gly Leu Leu Gly 1040 1045 1050 Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr Tyr Asn Asn 1055 1060 1065 Ile Arg Lys Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser Phe Ile 1070 1075 1080 Ser Lys Glu His Gly Trp Gln Glu Asn 1085 1090 15 1032 PRT Clostridium botulinum 15 Leu Val Ser Lys Phe Glu Asn Ser Val Lys Asn Ser Asn Lys Asn Tyr 1 5 10 15 Phe Thr Ile Asn Gly Leu Met Gly Tyr Tyr Phe Glu Asn Asp Phe Phe 20 25 30 Asn Leu Asn Ile Ile Ser Pro Thr Leu Asp Gly Asn Leu Thr Phe Ser 35 40 45 Lys Glu Asp Ile Asn Ser Ile Leu Gly Asn Lys Ile Ile Lys Ser Ala 50 55 60 Arg Trp Ile Gly Leu Ile Lys Pro Ser Ile Thr Gly Glu Tyr Ile Leu 65 70 75 80 Ser Thr Asn Ser Pro Asn Cys Arg Val Glu Leu Asn Gly Glu Ile Phe 85 90 95 Asn Leu Ser Leu Asn Thr Ser Asn Thr Val Asn Leu Ile Gln Gly Asn 100 105 110 Val Tyr Asp Ile Arg Ile Glu Gln Leu Met Ser Glu Asn Gln Leu Leu 115 120 125 Lys Asn Tyr Glu Gly Ile Lys Leu Tyr Trp Glu Thr Ser Asp Ile Ile 130 135 140 Lys Glu Ile Ile Pro Ser Glu Val Leu Leu Lys Pro Asn Tyr Ser Asn 145 150 155 160 Thr Asn Glu Lys Ser Lys Phe Ile Pro Asn Asn Thr Leu Phe Ser Asn 165 170 175 Ala Lys Leu Lys Ala Asn Ala Asn Arg Asp Thr Asp Arg Asp Gly Ile 180 185 190 Pro Asp Glu Trp Glu Ile Asn Gly Tyr Thr Val Met Asn Gln Lys Ala 195 200 205 Val Ala Trp Asp Asp Lys Phe Ala Ala Asn Gly Tyr Lys Lys Tyr Val 210 215 220 Ser Asn Pro Phe Lys Pro Cys Thr Ala Asn Asp Pro Tyr Thr Asp Phe 225 230 235 240 Glu Lys Val Ser Gly Gln Ile Asp Pro Ser Val Ser Met Val Ala Arg 245 250 255 Asp Pro Met Ile Ser Ala Tyr Pro Ile Val Gly Val Gln Met Glu Arg 260 265 270 Leu Val Val Ser Lys Ser Glu Thr Ile Thr Gly Asp Ser Thr Lys Ser 275 280 285 Met Ser Lys Ser Thr Ser His Ser Ser Thr Asn Ile Asn Thr Val Gly 290 295 300 Ala Glu Val Ser Gly Ser Leu Gln Leu Ala Gly Gly Ile Phe Pro Val 305 310 315 320 Phe Ser Met Ser Ala Ser Ala Asn Tyr Ser His Thr Trp Gln Asn Thr 325 330 335 Ser Thr Val Asp Asp Thr Thr Gly Glu Ser Phe Ser Gln Gly Leu Ser 340 345 350 Ile Asn Thr Gly Glu Ser Ala Tyr Ile Asn Pro Asn Ile Arg Tyr Tyr 355 360 365 Asn Thr Gly Thr Ala Pro Val Tyr Asn Val Thr Pro Thr Thr Thr Ile 370 375 380 Val Ile Asp Lys Gln Ser Val Ala Thr Ile Lys Gly Gln Glu Ser Leu 385 390 395 400 Ile Gly Asp Tyr Leu Asn Pro Gly Gly Thr Tyr Pro Ile Ile Gly Glu 405 410 415 Pro Pro Met Ala Leu Asn Thr Met Asp Gln Phe Ser Ser Arg Leu Ile 420 425 430 Pro Ile Asn Tyr Asn Gln Leu Lys Ser Ile Asp Asn Gly Gly Thr Val 435 440 445 Met Leu Ser Thr Ser Gln Phe Thr Gly Asn Phe Ala Lys Tyr Asn Ser 450 455 460 Asn Gly Asn Leu Val Thr Asp Gly Asn Asn Trp Gly Pro Tyr Leu Gly 465 470 475 480 Thr Ile Lys Ser Thr Thr Ala Ser Leu Thr Leu Ser Phe Ser Gly Gln 485 490 495 Thr Thr Gln Val Ala Val Val Ala Pro Asn Phe Ser Asp Pro Glu Asp 500 505 510 Lys Thr Pro Lys Leu Thr Leu Glu Gln Ala Leu Val Lys Ala Phe Ala 515 520 525 Leu Glu Lys Lys Asn Gly Lys Phe Tyr Phe His Gly Leu Glu Ile Ser 530 535 540 Lys Asn Glu Lys Ile Gln Val Phe Leu Asp Ser Asn Thr Asn Asn Asp 545 550 555 560 Phe Glu Asn Gln Leu Lys Asn Thr Ala Asp Lys Asp Ile Met His Cys 565 570 575 Ile Ile Lys Arg Asn Met Asn Ile Leu Val Lys Val Ile Thr Phe Lys 580 585 590 Glu Asn Ile Ser Ser Ile Asn Thr Met Ser Tyr Thr Asn Asp Lys Ile 595 600 605 Leu Ile Leu Tyr Phe Asn Lys Leu Tyr Lys Lys Ile Lys Asp Asn Ser 610 615 620 Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe Ile Asp Ile Ser Gly 625 630 635 640 Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asp Val Tyr Ile Tyr Ser Thr 645 650 655 Asn Arg Asn Gln Phe Gly Ile Tyr Ser Ser Lys Pro Ser Glu Val Asn 660 665 670 Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn Gly Arg Tyr Gln Asn Phe 675 680 685 Ser Ile Ser Phe Trp Val Arg Ile Pro Lys Tyr Phe Asn Lys Val Asn 690 695 700 Leu Asn Asn Glu Tyr Thr Ile Ile Asp Cys Ile Arg Asn Asn Asn Ser 705 710 715 720 Gly Trp Lys Ile Ser Leu Asn Tyr Asn Lys Ile Ile Trp Thr Leu Gln 725 730 735 Asp Thr Ala Gly Asn Asn Gln Lys Leu Val Phe Asn Tyr Thr Gln Met 740 745 750 Ile Ser Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile Thr 755 760 765 Asn Asn Arg Leu Gly Asn Ser Arg Ile Tyr Ile Asn Gly Asn Leu Ile 770 775 780 Asp Glu Lys Ser Ile Ser Asn Leu Gly Asp Ile His Val Ser Asp Asn 785 790 795 800 Ile Leu Phe Lys Ile Val Gly Cys Asn Asp Thr Arg Tyr Val Gly Ile 805 810 815 Arg Tyr Phe Lys Val Phe Asp Thr Glu Leu Gly Lys Thr Glu Ile Glu 820 825 830 Thr Leu Tyr Ser Asp Glu Pro Asp Pro Ser Ile Leu Lys Asp Phe Trp 835 840 845 Gly Asn Tyr Leu Leu Tyr Asn Lys Arg Tyr Tyr Leu Leu Asn Leu Leu 850 855 860 Arg Thr Asp Lys Ser Ile Thr Gln Asn Ser Asn Phe Leu Asn Ile Asn 865 870 875 880 Gln Gln Arg Gly Val Tyr Gln Lys Pro Asn Ile Phe Ser Asn Thr Arg 885 890 895 Leu Tyr Thr Gly Val Glu Val Ile Ile Arg Lys Asn Gly Ser Thr Asp 900 905 910 Ile Ser Asn Thr Asp Asn Phe Val Arg Lys Asn Asp Leu Ala Tyr Ile 915 920 925 Asn Val Val Asp Arg Asp Val Glu Tyr Arg Leu Tyr Ala Asp Ile Ser 930 935 940 Ile Ala Lys Pro Glu Lys Ile Ile Lys Leu Ile Arg Thr Ser Asn Ser 945 950 955 960 Asn Asn Ser Leu Gly Gln Ile Ile Val Met Asp Ser Ile Gly Asn Asn 965 970 975 Cys Thr Met Asn Phe Gln Asn Asn Asn Gly Gly Asn Ile Gly Leu Leu 980 985 990 Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr Tyr Asn Asn 995 1000 1005 Ile Arg Lys Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser Phe Ile 1010 1015 1020 Ser Lys Glu His Gly Trp Gln Glu Asn 1025 1030 16 1112 PRT Artificial Sequence synthetic construct 16 Leu Val Ser Lys Phe Glu Asn Ser Val Lys Asn Ser Asn Lys Asn Tyr 1 5 10 15 Phe Thr Ile Asn Gly Leu Met Gly Tyr Tyr Phe Glu Asn Asp Phe Phe 20 25 30 Asn Leu Asn Ile Ile Ser Pro Thr Leu Asp Gly Asn Leu Thr Phe Ser 35 40 45 Lys Glu Asp Ile Asn Ser Ile Leu Gly Asn Lys Ile Ile Lys Ser Ala 50 55 60 Arg Trp Ile Gly Leu Ile Lys Pro Ser Ile Thr Gly Glu Tyr Ile Leu 65 70 75 80 Ser Thr Asn Ser Pro Asn Cys Arg Val Glu Leu Asn Gly Glu Ile Phe 85 90 95 Asn Leu Ser Leu Asn Thr Ser Asn Thr Val Asn Leu Ile Gln Gly Asn 100 105 110 Val Tyr Asp Ile Arg Ile Glu Gln Leu Met Ser Glu Asn Gln Leu Leu 115 120 125 Lys Asn Tyr Glu Gly Ile Lys Leu Tyr Trp Glu Thr Ser Asp Ile Ile 130 135 140 Lys Glu Ile Ile Pro Ser Glu Val Leu Leu Lys Pro Asn Tyr Ser Asn 145 150 155 160 Thr Asn Glu Lys Ser Lys Phe Ile Pro Asn Asn Thr Leu Phe Ser Asn 165 170 175 Ala Lys Leu Lys Ala Asn Ala Asn Arg Asp Thr Asp Arg Asp Gly Ile 180 185 190 Pro Asp Glu Trp Glu Ile Asn Gly Tyr Thr Val Met Asn Gln Lys Ala 195 200 205 Val Ala Trp Asp Asp Lys Phe Ala Ala Asn Gly Tyr Lys Lys Tyr Val 210 215 220 Ser Asn Pro Phe Lys Pro Cys Thr Ala Asn Asp Pro Tyr Thr Asp Phe 225 230 235 240 Glu Lys Val Ser Gly Gln Ile Asp Pro Ser Val Ser Met Val Ala Arg 245 250 255 Asp Pro Met Ile Ser Ala Tyr Pro Ile Val Gly Val Gln Met Glu Arg 260 265 270 Leu Val Val Ser Lys Ser Glu Thr Ile Thr Gly Asp Ser Thr Lys Ser 275 280 285 Met Ser Lys Ser Thr Ser His Ser Ser Thr Asn Ile Asn Thr Val Gly 290 295 300 Ala Glu Val Ser Gly Ser Leu Gln Leu Ala Gly Gly Ile Phe Pro Val 305 310 315 320 Phe Ser Met Ser Ala Ser Ala Asn Tyr Ser His Thr Trp Gln Asn Thr 325 330 335 Ser Thr Val Asp Asp Thr Thr Gly Glu Ser Phe Ser Gln Gly Leu Ser 340 345 350 Ile Asn Thr Gly Glu Ser Ala Tyr Ile Asn Pro Asn Ile Arg Tyr Tyr 355 360 365 Asn Thr Gly Thr Ala Pro Val Tyr Asn Val Thr Pro Thr Thr Thr Ile 370 375 380 Val Ile Asp Lys Gln Ser Val Ala Thr Ile Lys Gly Gln Glu Ser Leu 385 390 395 400 Ile Gly Asp Tyr Leu Asn Pro Gly Gly Thr Tyr Pro Ile Ile Gly Glu 405 410 415 Pro Pro Met Ala Leu Asn Thr Met Asp Gln Phe Ser Ser Arg Leu Ile 420 425 430 Pro Ile Asn Tyr Asn Gln Leu Lys Ser Ile Asp Asn Gly Gly Thr Val 435 440 445 Met Leu Ser Thr Ser Gln Phe Thr Gly Asn Phe Ala Lys Tyr Asn Ser 450 455 460 Asn Gly Asn Leu Val Thr Asp Gly Asn Asn Trp Gly Pro Tyr Leu Gly 465 470 475 480 Thr Ile Lys Ser Thr Thr Ala Ser Leu Thr Leu Ser Phe Ser Gly Gln 485 490 495 Thr Thr Gln Val Ala Val Val Ala Pro Asn Phe Ser Asp Pro Glu Asp 500 505 510 Lys Thr Pro Lys Leu Thr Leu Glu Gln Ala Leu Val Lys Ala Phe Ala 515 520 525 Leu Glu Lys Lys Asn Gly Lys Phe Tyr Phe His Gly Leu Glu Ile Ser 530 535 540 Lys Asn Glu Lys Ile Gln Val Phe Leu Asp Ser Asn Thr Asn Asn Asp 545 550 555 560 Phe Glu Asn Gln Leu Lys Asn Thr Ala Asp Lys Asp Ile Met His Cys 565 570 575 Ile Ile Lys Arg Asn Met Asn Ile Leu Val Lys Val Ile Thr Phe Lys 580 585 590 Glu Asn Ile Ser Ser Ile Asn Ile Ile Asn Asp Thr Asn Phe Gly Val 595 600 605 Gln Ser Met Thr Gly Leu Ser Asn Arg Ser Lys Gly Gln Asp Gly Ile 610 615 620 Tyr Arg Ala Ala Thr Thr Ala Phe Ser Phe Lys Ser Lys Glu Leu Lys 625 630 635 640 Tyr Pro Glu Gly Arg Tyr Arg Met Arg Phe Val Ile Gln Ser Tyr Glu 645 650 655 Pro Phe Thr Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile 660 665 670 Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn 675 680 685 Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr 690 695 700 Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu 705 710 715 720 Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile 725 730 735 Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg 740 745 750 Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu 755 760 765 Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser 770 775 780 Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys 785 790 795 800 Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp 805 810 815 Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr 820 825 830 Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met 835 840 845 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn 850 855 860 Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser 865 870 875 880 Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile 885 890 895 Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe 900 905 910 Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val 915 920 925 Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met 930 935 940 Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr 945 950 955 960 Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr 965 970 975 Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys 980 985 990 Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys 995 1000 1005 Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly 1010 1015 1020 Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val 1025 1030 1035 Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr 1040 1045 1050 Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly 1055 1060 1065 Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn 1070 1075 1080 Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp 1085 1090 1095 Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp Leu Gln 1100 1105 1110 17 1052 PRT Artificial Sequence synthetic construct 17 Leu Val Ser Lys Phe Glu Asn Ser Val Lys Asn Ser Asn Lys Asn Tyr 1 5 10 15 Phe Thr Ile Asn Gly Leu Met Gly Tyr Tyr Phe Glu Asn Asp Phe Phe 20 25 30 Asn Leu Asn Ile Ile Ser Pro Thr Leu Asp Gly Asn Leu Thr Phe Ser 35 40 45 Lys Glu Asp Ile Asn Ser Ile Leu Gly Asn Lys Ile Ile Lys Ser Ala 50 55 60 Arg Trp Ile Gly Leu Ile Lys Pro Ser Ile Thr Gly Glu Tyr Ile Leu 65 70 75 80 Ser Thr Asn Ser Pro Asn Cys Arg Val Glu Leu Asn Gly Glu Ile Phe 85 90 95 Asn Leu Ser Leu Asn Thr Ser Asn Thr Val Asn Leu Ile Gln Gly Asn 100 105 110 Val Tyr Asp Ile Arg Ile Glu Gln Leu Met Ser Glu Asn Gln Leu Leu 115 120 125 Lys Asn Tyr Glu Gly Ile Lys Leu Tyr Trp Glu Thr Ser Asp Ile Ile 130 135 140 Lys Glu Ile Ile Pro Ser Glu Val Leu Leu Lys Pro Asn Tyr Ser Asn 145 150 155 160 Thr Asn Glu Lys Ser Lys Phe Ile Pro Asn Asn Thr Leu Phe Ser Asn 165 170 175 Ala Lys Leu Lys Ala Asn Ala Asn Arg Asp Thr Asp Arg Asp Gly Ile 180 185 190 Pro Asp Glu Trp Glu Ile Asn Gly Tyr Thr Val Met Asn Gln Lys Ala 195 200 205 Val Ala Trp Asp Asp Lys Phe Ala Ala Asn Gly Tyr Lys Lys Tyr Val 210 215 220 Ser Asn Pro Phe Lys Pro Cys Thr Ala Asn Asp Pro Tyr Thr Asp Phe 225 230 235 240 Glu Lys Val Ser Gly Gln Ile Asp Pro Ser Val Ser Met Val Ala Arg 245 250 255 Asp Pro Met Ile Ser Ala Tyr Pro Ile Val Gly Val Gln Met Glu Arg 260 265 270 Leu Val Val Ser Lys Ser Glu Thr Ile Thr Gly Asp Ser Thr Lys Ser 275 280 285 Met Ser Lys Ser Thr Ser His Ser Ser Thr Asn Ile Asn Thr Val Gly 290 295 300 Ala Glu Val Ser Gly Ser Leu Gln Leu Ala Gly Gly Ile Phe Pro Val 305 310 315 320 Phe Ser Met Ser Ala Ser Ala Asn Tyr Ser His Thr Trp Gln Asn Thr 325 330 335 Ser Thr Val Asp Asp Thr Thr Gly Glu Ser Phe Ser Gln Gly Leu Ser 340 345 350 Ile Asn Thr Gly Glu Ser Ala Tyr Ile Asn Pro Asn Ile Arg Tyr Tyr 355 360 365 Asn Thr Gly Thr Ala Pro Val Tyr Asn Val Thr Pro Thr Thr Thr Ile 370 375 380 Val Ile Asp Lys Gln Ser Val Ala Thr Ile Lys Gly Gln Glu Ser Leu 385 390 395 400 Ile Gly Asp Tyr Leu Asn Pro Gly Gly Thr Tyr Pro Ile Ile Gly Glu 405 410 415 Pro Pro Met Ala Leu Asn Thr Met Asp Gln Phe Ser Ser Arg Leu Ile 420 425 430 Pro Ile Asn Tyr Asn Gln Leu Lys Ser Ile Asp Asn Gly Gly Thr Val 435 440 445 Met Leu Ser Thr Ser Gln Phe Thr Gly Asn Phe Ala Lys Tyr Asn Ser 450 455 460 Asn Gly Asn Leu Val Thr Asp Gly Asn Asn Trp Gly Pro Tyr Leu Gly 465 470 475 480 Thr Ile Lys Ser Thr Thr Ala Ser Leu Thr Leu Ser Phe Ser Gly Gln 485 490 495 Thr Thr Gln Val Ala Val Val Ala Pro Asn Phe Ser Asp Pro Glu Asp 500 505 510 Lys Thr Pro Lys Leu Thr Leu Glu Gln Ala Leu Val Lys Ala Phe Ala 515 520 525 Leu Glu Lys Lys Asn Gly Lys Phe Tyr Phe His Gly Leu Glu Ile Ser 530 535 540 Lys Asn Glu Lys Ile Gln Val Phe Leu Asp Ser Asn Thr Asn Asn Asp 545 550 555 560 Phe Glu Asn Gln Leu Lys Asn Thr Ala Asp Lys Asp Ile Met His Cys 565 570 575 Ile Ile Lys Arg Asn Met Asn Ile Leu Val Lys Val Ile Thr Phe Lys 580 585 590 Glu Asn Ile Ser Ser Ile Asn Lys Asn Leu Asp Cys Trp Val Asp Asn 595 600 605 Glu Glu Asp Ile Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu 610 615 620 Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser 625 630 635 640 Val Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys 645 650 655 Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys 660 665 670 Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser 675 680 685 Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr 690 695 700 Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu 705 710 715 720 Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile 725 730 735 Trp Thr Leu Lys Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg 740 745 750 Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe 755 760 765 Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn 770 775 780 Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg 785 790 795 800 Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn 805 810 815 Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn 820 825 830 Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 835 840 845 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr 850 855 860 Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile 865 870 875 880 Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys 885 890 895 Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile 900 905 910 Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly 915 920 925 Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val 930 935 940 Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu 945 950 955 960 Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu 965 970 975 Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu 980 985 990 Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly 995 1000 1005 Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn 1010 1015 1020 Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp 1025 1030 1035 Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp Leu Gln 1040 1045 1050 18 10 PRT Artificial Sequence synthetic construct 18 Cys Gly Ile Glu Gly Arg Ala Pro Gly Pro 1 5 10 

1. A composition comprising a therapeutic agent linked to a polypeptide, wherein the polypeptide is a non-toxic polypeptide, for delivery of a therapeutic agent to a neuronal cell, comprising:— a binding domain that binds to the neuronal cell and is comprised of or derived from clostridial heavy chain fragments or modified clostridial heavy chain fragments, and a translocation domain that translocates the therapeutic agent into the neuronal cell, wherein the translocation domain is not a H_(N) domain of a clostridial toxin and is not a fragment or derivative of a H_(N) domain of a clostridial toxin and wherein the therapeutic agent is for reduction of neuronal damage after ischemia/reperfusion or is for promotion of neuronal growth after damage.
 2. A composition according to claim 1, wherein the translocation domain is further a non-aggregating translocation domain as measured by size in physiological buffers.
 3. A composition according to claim 1 or 2, wherein the translocation domain is further selected from (1) a H_(N) domain of a diphtheria toxin, (2) a fragment or derivative of (1) that substantially retains the translocating activity of the H_(N) domain of a diphtheria toxin, (3) a fusogenic peptide, (4) a membrane disrupting peptide, and (5) translocating fragments and derivatives of (3) and (4).
 4. A composition according to any of claims 1 to 3, wherein the polypeptide has the binding specificity of tetanus toxin and reduced affinity to neutralising antibodies to tetanus toxin compared with the affinity to such antibodies of native tetanus toxin heavy chain.
 5. A composition comprising a therapeutic agent linked to a polypeptide, wherein the polypeptide is for delivery of a therapeutic agent to a neuronal cell, comprising:— a binding domain that binds to the neuronal cell and is comprised of or derived from clostridial heavy chain fragments or modified clostridial heavy chain fragments, and a translocation domain that translocates the therapeutic agent into the neuronal cell, wherein the translocation domain is a non-aggregating translocation domain as measured by its size in physiological buffer and wherein the therapeutic agent is for reduction of neuronal damage after ischemia/reperfusion or is for promotion of neuronal growth after damage.
 6. A composition according to claim 5, wherein the translocation domain is further selected from (1) a H_(N) domain of a diphtheria toxin, (2) a fragment or derivative of (1) that substantially retains the translocating activity of the H_(N) domain of a diphtheria toxin, (3) a fusogenic peptide, (4) a membrane disrupting peptide, and (5) translocating fragments and derivatives of (3) and (4).
 7. A composition according to claim 5 or 6, wherein the polypeptide has reduced affinity to neutralising antibodies to tetanus toxin compared with the affinity to such antibodies of native tetanus toxin heavy chain.
 8. A composition comprising a therapeutic agent linked to a polypeptide, wherein the polypeptide is for delivery of a therapeutic agent to a neuronal cell, comprising:— a binding domain that binds to the neuronal cell and is comprised of or derived from clostridial heavy chain fragments or modified clostridial heavy chain fragments, and a translocation domain that translocates the therapeutic agent into the neuronal cell, wherein the translocation domain is selected from (1) a H_(N) domain of a diphtheria toxin, (2) a fragment or derivative of (1) that substantially retains the translocating activity of the H_(N) domain of a diphtheria toxin, (3) a fusogenic peptide, (4) a membrane disrupting peptide, and (5) translocating fragments and derivatives of (3) and (4) and wherein the therapeutic agent is for reduction of neuronal damage after ischemia/reperfusion or is for promotion of neuronal growth after damage.
 9. A composition according to claim 8, wherein the polypeptide has reduced affinity to neutralising antibodies to tetanus toxin compared with the affinity to such antibodies of native tetanus toxin.
 10. A composition comprising a therapeutic agent linked to a polypeptide, wherein the polypeptide is for delivery of a therapeutic agent to a neuronal cell, comprising:— a binding domain that binds to the neuronal cell and is comprised of or derived from clostridial heavy chain fragments or modified clostridial heavy chain fragments, and a translocation domain that translocates the therapeutic agent into the neuronal cell, wherein the polypeptide has reduced affinity to neutralising antibodies to tetanus toxin compared with the affinity to such antibodies of native tetanus toxin and wherein the therapeutic agent is for reduction of neuronal damage after ischemia/reperfusion or is for promotion of neuronal growth after damage.
 11. A composition according to any previous claim wherein the binding domain comprises a botulinum H_(C) domain.
 12. A composition according to any of claims 1 to 10 wherein the binding domain comprises a tetanus H_(C) domain.
 13. A composition according to any of claims 1 to 10 wherein the binding domain comprises a hybrid of a botulinum H_(C) domain and a tetanus H_(C) domain.
 14. A composition according to claim 13, based on a botulinum type B fragment (residues 859-1291) or type A fragment (residues 872-1296) or type F fragment (residues 862-1278).
 15. A composition according to claim 13, based on a botulinum type B fragment (residues 859-1291) by replacing residues 1093-1285 with the homologous sequence from tetanus toxin.
 16. A composition according to claim 13, based on a botulinum type A fragment (residues 872-1296) by replacing residues 1103-1296 with the homologous sequence from tetanus toxin.
 17. A composition according to claim 13, based on a botulinum type F fragment (residues 862-1278) by replacing residues 1097-1273 with the homologous sequence from tetanus toxin.
 18. A composition according to any of claims 1 to 10 comprising a tetanus H_(C) domain and a diphtheria H_(N) domain.
 19. A composition according to any of claims 1 to 10 comprising a botulinum H_(C) domain and diphtheria H_(N) domain.
 20. A composition according to any of claims 1-19, wherein the therapeutic substance is chemically bound to said polypeptide.
 21. A composition according to any of claims 1-20 wherein the therapeutic substance is linked to a translocation domain of said polypeptide.
 22. A composition according to any of claims 1 to 21 wherein the therapeutic substance is an enzyme, growth factor, protein or peptide.
 23. A composition according to any of claims 21 to 22 wherein the therapeutic agent is produced as a fusion protein by recombinant technology methods.
 24. A composition according to any of claims 1 to 23 wherein the therapeutic agent is a nucleic acid useful as a gene therapeutic agent or an antisense drug.
 25. A composition according to claim 24 in which the nucleic acid is contained within a liposome or is condensed via a peptide or protein, or by condensation using a chemical coupling agent.
 26. A composition according to claim 24 in which the nucleic acid is present in the form of a recombinant virus.
 27. A composition according to claim 24 in which the virus has an altered tropism and is capable of transducing cells via a clostridial toxin receptor.
 28. A method of manufacture of a composition according to any of claims 1 to 19, comprising expressing in a host cell a nucleic acid encoding the composition.
 29. Use of a composition according to any of claims 1 to 27 in the treatment of a disease state associated with neuronal cells. 